Novel anti-pd-1 antibodies

ABSTRACT

The present invention provides novel anti-PD-1 antibodies that specifically bind to cell surface PD-1. Also provided are the nucleic acid molecules encoding the anti-PD-1 antibodies, expression vectors and host cells used for the expression of the anti-PD-1 antibodies. The invention further provides the methods for producing the anti-PD-1 antibodies and the use thereof.

SEQUENCE LISTING

The instant application contains a sequence listing and is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This application generally relates to antibodies. More specifically, theapplication relates to single-domain antibodies that specifically bindto PD-1, a method for preparing the same, and the use thereof.

BACKGROUND OF THE INVENTION

Increasing evidences from preclinical and clinical results have shownthat targeting immune checkpoints is becoming the most promisingapproach to treat patients with cancers. The protein Programmed Death 1(PD-1), an inhibitory member of the immunoglobulin super-family withhomology to CD28, is expressed on T cells, activated B cells, andmyeloid cells (Agata et al, supra; Okazaki et al (2002) Curr. Opin.Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8) andhas a critical role in regulating stimulatory and inhibitory signals inthe immune system (Okazaki, Taku et al. 2007 International Immunology19:813-824). PD-1 was discovered through screening for differentialexpression in apoptotic cells (Ishida et al (1992) EMBO J 11:3887-95). pThe PD-1 is a type I transmembrane protein that is part of the Ig genesuperfamily (Agata et al. (1996) bit Immunol 8:765-72) and the structureof PD-1 consists of one immunoglobulin variable-like extracellulardomain and a cytoplasmic domain containing an immunoreceptortyrosine-based inhibitory motif (ITIM) and an immunoreceptortyrosine-based switch motif (ITSM). Although structurally similar toCTLA-4, PD-1 lacks the MYPPPY motif that is critical for B7-1 and B7-2binding. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2(B7-DC, CD273), which are cell surface expressed members of the B7family (Freeman et al (2000) J Exp Med 192: 1027-34; Latchman et al(2001) Nat Immunol 2:261-8; Carter et al (2002) Eur J Immunol32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, butdo not bind to other CD28 family members.

PD-1, as one of the immune-checkpoint proteins, is an inhibitory memberof the CD28 family expressed on activated B cells, T cells, and myeloidcells (Agata et al, supra; Okazaki et al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8), and plays a majorrole in limiting the activity of T cells that provide a major immuneresistance mechanism by which tumor cells escaped immune surveillance.PD-1 induces a state of anergy or unresponsiveness in T cells, resultingin the cells being unable to produce optimal levels of effectorcytokines. PD-1 may also induce apoptosis in T cells via its ability toinhibit survival signals. PD-1 deficient animals develop variousautoimmune phenotypes, including autoimmune cardiomyopathy and alupus-like syndrome with arthritis and nephritis (Nishimura et al.(1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22).Additionally, PD-1 has been found to play a role in autoimmuneencephalomyelitis, systemic lupus erythematosus, graft-versus-hostdisease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al.(2003) J Exp Med 198:71-78: Prokunina and Alarcon-Riquelme (2004) HumMoI Genet 13:R143; Nielsen et al. (2004) Lupus 11:510). In a murine Bcell tumor line, the ITSM of PD-1 was shown to be essential to blockBCR-mediated Ca2+-flux and tyrosine phosphorylation of downstreameffector molecules (Okazaki et al. (2001) PNAS 98: 13866-71).

The interaction of PD-1 expressed on activated T cells, and PD-L1expressed on tumor cells negatively regulates immune response and dampsanti-tumor immunity. PD-L1 is abundant in a variety of human cancers(Dong et al (2002) Nat. Med 8:787-9). Expression of PD-L1 on tumors iscorrelated with reduced survival in esophageal, pancreatic and othertypes of cancers, highlighting this pathway as a promising target fortumor immunotherapy. Several groups have shown that the PD-1-PD-L1interaction exacerbates disease, resulting in a decrease in tumorinfiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and immune evasion by the cancerous cells (Dong et al.(2003) J. MoI. Med. 81:281-7; Blank et al. (2005) Cancer Immunol.Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res.10:5094-100). Immune suppression can be reversed by inhibiting the localinteraction of PD-1 with PD-L1, and the effect is additive when theinteraction of PD-1 with PD-L2 is blocked as well.

A single-domain antibody (sdAb) is an antibody consisting of a singlemonomeric variable antibody domain. Like a whole antibody, it is able tobind selectively to a specific antigen. Single-domain antibodies aremuch smaller than common antibodies which are composed of two heavyprotein chains and two light chains. The first single-domain antibodieswere engineered from heavy-chain antibodies found in camelids(Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C,Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodiesdevoid of light chains. Nature 363(6428):446-448.); these are called VHHfragments. Currently, most research into single-domain antibodies isbased on heavy chain variable domains.

Single-domain antibodies have many advantages. For instance, theygenerally display high solubility and stability and can also be readilyproduced in yeast, plant, and mammalian cells (Harmsen M M, De Haard H J(2007) Properties, production, and applications of camelid single-domainantibody fragments. Appl Microbiol Biotechnol 77(1):13-22.). Further,they have good thermal stability and good tissue penetration. And theyare also cost efficient in production. The advantages of single-domainantibodies make them suitable for various biotechnological andtherapeutic applications. For instance, they are useful in the treatmentof diseases, including but not limited to cancer, infectious,inflammatory and neurodegenerative diseases.

Although antibodies against PD-1 are been developed, there are stillspaces for improvement for antibody against PD-1 as a therapeutic agent.Further, it is worth noting that there are few single-domain antibodiesagainst PD-1 currently. Accordingly, there is desire in the art todevelop novel anti-PD-1 antibodies, particularly single-domainantibodies against PD-1.

SUMMARY OF THE INVENTION

These and other objectives are provided for by the present inventionwhich, in a broad sense, is directed to novel compounds, methods,compositions and articles of manufacture that provide antibodies withimproved efficacy. The benefits provided by the present invention arebroadly applicable in the field of antibody therapeutics and diagnosticsand may be used in conjunction with antibodies that react with a varietyof targets. The present invention provides anti-PD-1 antibodies,preferably single-domain antibodies. It also provides methods ofpreparing the antibodies, nucleic acid molecules encoding the anti-PD-1antibodies, expression vectors and host cells used for the expression ofanti-PD-1 antibodies. The antibodies of the invention provide methodsfor treating or preventing conditions associated with PD-1.

In some aspects, the invention is directed to a PD-1 binding molecule.

In some embodiments, the PD-1 binding molecule has one or more of thefollowing properties:

(a) binds human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(b) inhibits binding of PD-L1/2 to PD-1;

(c) induces production of IFN-γ in CD4+T cells;

(d) does not substantially bind to human CD28, CTLA-4, ICOS and BTL;

(e) has no cross-reactivity with human PD-1, but has cross-reactivitywith mouse PD-1; and

(f) is stable at least 60° C. as measured by DSF assay.

In some embodiments, the PD-1 binding molecule comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 comprises an aminoacid sequence which is at least 90% identical to SEQ ID NO: 1, CDR2comprises an amino acid sequence which is at least 90% identical to SEQID NO: 2, and CDR3 comprises an amino acid sequence which is at least80% identical to SEQ ID NO: 3.

In some embodiments, the PD-1 binding molecule comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 differs in aminoacid sequence from SEQ ID NO: 1 by an amino acid addition, deletion orsubstitution of not more than 2 amino acids; CDR2 differs in amino acidsequence from SEQ ID NO: 2 by an amino acid addition, deletion orsubstitution of not more than 2 amino acids; and/or CDR3 differs inamino acid sequence from SEQ ID NO: 3 by an amino acid addition,deletion or substitution of not more than 2 amino acids.

In some embodiments, the PD-1 binding molecule comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises CDR1, CDR2 and CDR3, and wherein CDR1, CDR2 and CDR3 areselected from the group comprising:

(a) CDR1 which is represented by DSIX₁SX₂VNMG, wherein X₁=D or Q, andX₂=M or L;

(b) CDR2 which is represented by LIAX₃YITHYADFVKG, wherein X₃=N, T, Y, Ror W;

(c) CDR3 which is represented by RX₄IX₅X₆DY, wherein X₄=N or S, X₅=I, Ror Y, and X₆=V or E.

In some embodiments, the PD-1 binding molecule comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises CDR1, CDR2 and CDR3, and wherein CDR1, CDR2 and CDR3 areselected from the group comprising:

(a) CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 withan amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 3;

(b) CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, CDR2 withan amino acid sequence as shown in SEQ ID NO: 5, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 6;

(c) CDR1 with an amino acid sequence as shown in SEQ ID NO: 7, CDR2 withan amino acid sequence as shown in SEQ ID NO: 8, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 9;

(d) CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, CDR2with an amino acid sequence as shown in SEQ ID NO: 11, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 12;

(e) CDR1 with an amino acid sequence as shown in SEQ ID NO: 13, CDR2with an amino acid sequence as shown in SEQ ID NO: 14, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 15;

(f) CDR1 with an amino acid sequence as shown in SEQ ID NO: 16, CDR2with an amino acid sequence as shown in SEQ ID NO: 17, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 18;

(g) CDR1 with an amino acid sequence as shown in SEQ ID NO: 19, CDR2with an amino acid sequence as shown in SEQ ID NO: 20, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 21;

(h) CDR1 with an amino acid sequence as shown in SEQ ID NO: 22, CDR2with an amino acid sequence as shown in SEQ ID NO: 23, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 24;

(i) CDR1 with an amino acid sequence as shown in SEQ ID NO: 25, CDR2with an amino acid sequence as shown in SEQ ID NO: 26, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 27;

(j) CDR1 with an amino acid sequence as shown in SEQ ID NO: 28, CDR2with an amino acid sequence as shown in SEQ ID NO: 29, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 30;

(k) CDR1 with an amino acid sequence as shown in SEQ ID NO: 31, CDR2with an amino acid sequence as shown in SEQ ID NO: 32, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 33; and

(l) CDR1 with an amino acid sequence as shown in SEQ ID NO: 34, CDR2with an amino acid sequence as shown in SEQ ID NO: 35, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 36.

In some embodiments, the PD-1 binding molecule comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises

(A) the amino acid sequence shown in any of SEQ ID NOs: 37-49;

(B) an amino acid sequence which is at least 85%, at least 90%, or atleast 95% identical to any of SEQ ID NOs: 37-49; or

(C) an amino acid sequence with addition, deletion and/or substitutionof one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) aminoacids compared with any of SEQ ID NOs: 37-49.

In some aspects, the invention is directed to an isolated nucleic acidmolecule, comprising a nucleic acid sequence encoding PD-1 bindingmolecule as disclosed herein.

In some aspects, the invention is directed to an expression vectorcomprising the nucleic acid molecule encoding the PD-1 binding moleculeas disclosed herein.

In some aspects, the invention is directed to a host cell comprising theexpression vector as disclosed herein.

In some aspects, the invention is directed to a pharmaceuticalcomposition comprising at least one PD-1 binding molecule as disclosedherein and a pharmaceutically acceptable carrier.

In some aspects, the invention is directed to a method for preparing thePD-1 binding molecule which comprises expressing the PD-1 bindingmolecule in the host cell and isolating the PD-1 binding molecule fromthe host cell.

In some aspects, the invention is directed to a method for inhibiting orblocking the binding of PD-L1 to PD-1 in a subject, comprising:administering a therapeutically effective amount of the PD-1 bindingmolecule as disclosed herein to the subject.

In some aspects, the invention is directed to a method for inhibiting orblocking the binding of PD-L2 to PD-1 in a subject, comprising:administering a therapeutically effective amount of the PD-1 bindingmolecule as disclosed herein to the subject.

In some aspects, the invention is directed to a method of treating acondition associated with PD-1 in a subject, comprising: administering atherapeutically effective amount of the PD-1 binding molecule asdisclosed herein to the subject.

In some aspects, the invention is directed to a method of treating acondition in a subject that would benefit from upregulation of immuneresponse, comprising administering a therapeutically effective amount ofthe PD-1 binding molecule as disclosed herein to the subject.

In some aspects, the invention is directed to the use of the PD-1binding molecule as disclosed herein in the manufacture of a medicamentfor treating or preventing proliferative disorders such as cancers.

In some aspects, the invention is directed to the use of PD-1 bindingmolecule as disclosed herein in the manufacture of a medicament fortreating or preventing a condition that would benefit from upregulationof immune response.

In some aspects, the invention is directed to kits or devices andassociated methods that employ the PD-1 binding molecule as disclosedherein, and pharmaceutical compositions as disclosed herein, which areuseful for the treatment of proliferative disorders such as cancer. Tothis end the present invention preferably provides an article ofmanufacture useful for treating such disorders comprising a receptaclecontaining the PD-1 binding molecule as disclosed herein andinstructional materials for using the PD-1 binding molecule as disclosedherein to treat, ameliorate or prevent a proliferative disorder orprogression or recurrence thereof. In selected embodiments, the devicesand associated methods will comprise the step of contacting at least onecirculating tumor cell with the PD-1 binding molecule as disclosedherein.

The foregoing is a summary and thus contains, by necessity,simplifications, generalizations, and omissions of detail; consequently,those skilled in the art will appreciate that the summary isillustrative only and is not intended to be in any way limiting. Otheraspects, features, and advantages of the methods, compositions and/ordevices and/or other subject matter described herein will becomeapparent in the teachings set forth herein. The summary is provided tointroduce a selection of concepts in a simplified form that are furtherdescribed below in the Detailed Description. This summary is notintended to identify key features or essential features of the claimedsubject matter, nor is it intended to be used as an aid in determiningthe scope of the claimed subject matter. Further, the contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein in entirety by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding of anti-PD-1 antibodies to cell surface humanPD-1, expressed by MFI (Median Fluorescence Intensity) and measured byFACS.

FIG. 2 shows the binding of anti-PD-1 antibodies to cell surface mousePD-1, expressed by MFI and measured by FACS.

FIG. 3 shows the binding of anti-PD-1 antibodies to cell surfacecynomolgus PD-1, expressed by MFI and measured by FACS.

FIG. 4 shows the human PD1/PD-L1 blocking as measured by FACS.

FIG. 5 shows the mouse PD1/PD-L1 blocking as measured by FACS.

FIG. 6 shows the human PD1/PD-L2 blocking as measured by ELISA.

FIG. 7 shows the cynomolgus PD1/PD-L1 blocking as measured by ELISA.

FIG. 8 shows cross-reactivity to human PD-1, CD28, CTLA-4, ICOS and BTLAas measured by ELISA.

FIG. 9 shows epitope binning of the antibody AP17R1-2H2 (FIGS. 9A and9B) and the antibody W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) (FIGS. 9Cand 9D) against benchmark antibodies BMK1 and BMK3, respectively.

FIG. 10 shows the effects of anti-PD-1 antibodies on human allogeneicmixed lymphocyte reaction (Allo-MLR), as measured by ELISA and reflectedby the level of IL-2 (FIG. 10A) and IFN-γ (FIG. 10B).

FIG. 11 shows that the effects of anti-PD-1 antibodies on humanautologous MLR (Auto-MLR), as reflected by the level of IFN-y production(FIG. 11A) and T cell proliferation (FIG. 11B).

FIG. 12 shows the effects of anti-PD-1 antibodies on human Treg MLR, asreflected by the level of IFN-γ production (FIG. 12A) and T cellproliferation (FIG. 12B).

FIG. 13 shows the ADCC assays of the anti-PD-1 antibodies.

FIG. 14 shows the results of thermal stability test by DSF (DifferentialScanning Fluorimetry) assay.

FIG. 15 shows the stability of theW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) in human serum at 37° C.measured by target binding of serum treated samples by FACS

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodimentsillustrated. Moreover, any section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Morespecifically, as used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “a cell”includes mixtures of cells, and the like. In this application, the useof “or” means “and/or” unless stated otherwise. Furthermore, the use ofthe term “comprising,” as well as other forms, such as “comprises” and“comprised”, is not limiting. In addition, ranges provided in thespecification and appended claims include both end points and all pointsbetween the end points.

Generally, nomenclature used in connection with, and techniques of, celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Abbas et al., Cellular and Molecular Immunology,6^(th) ed., W.B. Saunders Company (2010); Sambrook J. & Russell D.Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlowand Lane Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al.,Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Thenomenclature used in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are well knownand commonly used in the art. Moreover, any section headings used hereinare for organizational purposes only and are not to be construed aslimiting the subject matter described.

Definitions

In order to better understand the invention, the definitions andexplanations of the relevant terms are provided as follows.

The term “antibody” or “Ab”, as used herein, generally refers to anyform of antibody that exhibits the desired biological or bindingactivity. It covers, but is not limited to, humanized antibodies, fullyhuman antibodies, chimeric antibodies and single-domain antibodies. Anantibody may comprise heavy chain(s) and light chain(s). Heavy chainsmay be classified into μ, δ, γ, α and ε, which define isotypes of anantibody as IgM, IgD, IgG, IgA and IgE, respectively. Each heavy chainconsists of a heavy chain variable region (V_(H)) and a heavy chainconstant region (C_(H)). A heavy chain constant region consists of 3domains (C_(H)1, C_(H)2 and C_(H)3). Each light chain consists of alight chain variable region (V_(L)) and a light chain constant region(C_(L)). V_(H) and V_(L) region can further be divided intohypervariable regions (called complementary determining regions (CDR)),which are interspaced by relatively conservative regions (calledframework region (FR)). Each V_(H) and V_(L) consists of 3 CDRs and 4FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 fromN-terminal to C-terminal. Distribution of amino acids in various regionsor domains follows the definition in Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917;Chothia et al., (1989) Nature 342:878-883. Antibodies may be ofdifferent antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 orIgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

The term “humanized antibody”, as used herein, refers to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody”, as used herein, refers to antibodies inwhich the variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The term “PD-1”, as used herein, refers programmed cell death protein,which belongs to the superfamily of immunoglobulin and functions asco-inhibitory receptor to negatively regulate the immune system. PD-1 isa member of the CD28/CTLA-4 family, and has two known ligands includingPD-L1 and PD-L2. Alternative names or synonyms for PD-1 include PDCD1,PD1, CD279 and SLEB2, et al. Representative amino acid sequence of humanPD-1 is disclosed under the NCBI accession number: NP_005009.2, and therepresentative nucleic acid sequence encoding the human PD-1 is shownunder the NCBI accession number: NM_005018.2.

The term “PD-L1”, as used herein, refers to programmed cell death ligand1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192:1027). Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1,B7H1, CD274 and B7-H, et al. Representative amino acid sequence of humanPD-L1 is disclosed under the NCBI accession number: NP_054862.1, and therepresentative nucleic acid sequence encoding the human PD-L1 is shownunder the NCBI accession number: NM_014143.3. PD-L1 is expressed inplacenta, spleen, lymph nodes, thymus, heart, fetal liver, and is alsofound on many tumor or cancer cells. PD-L1 binds to its receptor PD-1 orB7-1, which is expressed on activated T cells, B cells and myeloidcells. The binding of PD-L1 and its receptor induces signal transductionto suppress TCR-mediated activation of cytokine production and T cellproliferation. Accordingly, PD-L1 plays a major role in suppressingimmune system during particular events such as pregnancy, autoimmunediseases, tissue allografts, and is believed to allow tumor or cancercells to circumvent the immunological checkpoint and evade the immuneresponse.

The term “PD-L2”, as used herein, refers to programmed cell death ligand2. Alternative names or synonyms for PD-L2 include PDCD1L2, PDL2, B7-DC,Btdc and CD273, et al. Representative amino acid sequence of human PD-L2is disclosed under the NCBI accession number: NP_079515.2.

The term “Anti-PD-1 antibody”, as used herein, refers to an antibodythat is capable of specific binding to PD-1 (e.g. human, monkey ormonkey PD-1). It is advantage that the Anti-PD-1 antibody specificallybinds to PD-1 with an affinity which is sufficient to provide fordiagnostic and/or therapeutic use.

The term “Ka”, as used herein, is intended to refer to the associationrate of a particular antibody-antigen interaction, whereas the term “Kd”as used herein, is intended to refer to the dissociation rate of aparticular antibody-antigen interaction. Kd values for antibodies can bedetermined using methods well established in the art. The term “K_(D)”as used herein, is intended to refer to the dissociation constant of aparticular antibody-antigen interaction, which is obtained from theratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molarconcentration (M). A preferred method for determining the Kd of anantibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

The term “specific binding” or “specifically binds” as used hereinrefers to a non-random binding reaction between two molecules, such asfor example between an antibody and an antigen.

The ability to “inhibit binding”, “block binding” or “compete for thesame epitope”, as used herein, refers to the ability of an antibody toinhibit the binding interaction between two molecules (e.g. human PD-1and an anti-PD-1 antibody) to any detectable degree. In someembodiments, an antibody that blocks binding between two moleculesinhibits the binding interaction between the two molecules by at least50%. In some embodiments, this inhibition may be greater than 60%,greater than 70%, greater than 80%, or greater than 90%.

The term “high affinity” for an IgG antibody, as used herein, refers toan antibody having a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ Mor less, even more preferably 1×10⁻⁸ M or less, even more preferably5×10⁻⁹ M or less and even more preferably 1×10⁻⁹ M or less for a targetantigen.

The term “EC₅₀”, as used herein, which is also termed as “half maximaleffective concentration” refers to the concentration of a drug, antibodyor toxicant which induces a response halfway between the baseline andmaximum after a specified exposure time. In the context of theapplication, EC₅₀ is expressed in the unit of “nM”.

The term “epitope”, as used herein, refers to a portion on antigen thatan immunoglobulin or antibody specifically binds to. “Epitope” is alsoknown as “antigenic determinant”. Epitope or antigenic determinantgenerally consists of chemically active surface groups of a moleculesuch as amino acids, carbohydrates or sugar side chains, and generallyhas a specific three-dimensional structure and a specific chargecharacteristic. For example, an epitope generally comprises at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive ornon-consecutive amino acids in a unique steric conformation, which maybe “linear” or “conformational”. See, for example, Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996). In a linear epitope, all the interaction sites between a proteinand an interaction molecule (e.g., an antibody) are present linearlyalong the primary amino acid sequence of the protein. In aconformational epitope, the interaction sites span over amino acidresidues that are separate from each other in a protein. Antibodies maybe screened depending on competitiveness of binding to the same epitopeby conventional techniques known by a person skilled in the art. Forexample, study on competition or cross-competition may be conducted toobtain antibodies that compete or cross-compete with each other forbinding to antigens (e.g. RSV fusion protein). High-throughput methodsfor obtaining antibodies binding to the same epitope, which are based ontheir cross-competition, are described in an international patentapplication WO 03/48731.

The term “isolated”, as used herein, refers to a state obtained fromnatural state by artificial means. If a certain “isolated” substance orcomponent is present in nature, it is possible because its naturalenvironment changes, or the substance is isolated from naturalenvironment, or both. For example, a certain un-isolated polynucleotideor polypeptide naturally exists in a certain living animal body, and thesame polynucleotide or polypeptide with a high purity isolated from sucha natural state is called isolated polynucleotide or polypeptide. Theterm “isolated” excludes neither the mixed artificial or synthesizedsubstance nor other impure substances that do not affect the activity ofthe isolated substance.

The term “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds a PD-1 protein is substantially free of antibodies thatspecifically bind antigens other than PD-1 proteins). An isolatedantibody that specifically binds a human PD-1 protein may, however, havecross-reactivity to other antigens, such as PD-1 proteins from otherspecies. Moreover, an isolated antibody can be substantially free ofother cellular material and/or chemicals.

The term “vector”, as used herein, refers to a nucleic acid vehiclewhich can have a polynucleotide inserted therein. When the vector allowsfor the expression of the protein encoded by the polynucleotide insertedtherein, the vector is called an expression vector. The vector can havethe carried genetic material elements expressed in a host cell bytransformation, transduction, or transfection into the host cell.Vectors are well known by a person skilled in the art, including, butnot limited to plasmids, phages, cosmids, artificial chromosome such asyeast artificial chromosome (YAC), bacterial artificial chromosome (BAC)or P1-derived artificial chromosome (PAC); phage such as λ phage or M13phage and animal virus. The animal viruses that can be used as vectors,include, but are not limited to, retrovirus (including lentivirus),adenovirus, adeno-associated virus, herpes virus (such as herpes simplexvirus), pox virus, baculovirus, papillomavirus, papova virus (such asSV40). A vector may comprise multiple elements for controllingexpression, including, but not limited to, a promoter sequence, atranscription initiation sequence, an enhancer sequence, a selectionelement and a reporter gene. In addition, a vector may comprise originof replication.

The term “host cell”, as used herein, refers to a cell into which avector can be introduced, including, but not limited to, prokaryoticcell such as E. coli or Bacillus subtilis, fungal cell such as yeastcell or Aspergillus, insect cell such as S2 Drosophila cell or Sf9, andanimal cell such as fibroblast, CHO cell, COS cell, NSO cell, HeLa cell,BHK cell, HEK 293 cell or human cell.

The term “T cell”, as used herein, includes CD4+ T cells, CD8+ T cells,T helper 1 type T cells, T helper 2 type T cells, T helper 17 type Tcells and inhibitory T cells.

The term “identity”, as used herein, refers to a relationship betweenthe sequences of two or more polypeptide molecules or two or morenucleic acid molecules, as determined by aligning and comparing thesequences. “Percent identity” means the percent of identical residuesbetween the amino acids or nucleotides in the compared molecules and iscalculated based on the size of the smallest of the molecules beingcompared. For these calculations, gaps in alignments (if any) arepreferably addressed by a particular mathematical model or computerprogram (i.e., an “algorithm”). Methods that can be used to calculatethe identity of the aligned nucleic acids or polypeptides include thosedescribed in Computational Molecular Biology, (Lesk, A. M., ed.), 1988,New York: Oxford University Press; Biocomputing Informatics and GenomeProjects, (Smith, D. W., ed.), 1993, New York: Academic Press; ComputerAnalysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G.,eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, SequenceAnalysis in Molecular Biology, New York: Academic Press; SequenceAnalysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York:M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math.48:1073.

The term “immunogenicity”, as used herein, refers to ability ofstimulating the formation of specific antibodies or sensitizedlymphocytes in organisms. It not only refers to the property of anantigen to stimulate a specific immunocyte to activate, proliferate anddifferentiate so as to finally generate immunologic effector substancesuch as antibody and sensitized lymphocyte, but also refers to thespecific immune response that antibody or sensitized T lymphocyte can beformed in immune system of an organism after stimulating the organismwith an antigen. Immunogenicity is the most important property of anantigen. Whether an antigen can successfully induce the generation of animmune response in a host depends on three factors, properties of anantigen, reactivity of a host, and immunization means.

The term “transfection” or “transfect”, as used herein, refers to theprocess by which nucleic acids are introduced into eukaryotic cells,particularly mammalian cells. Protocols and techniques for transfectioninclude but not limited to lipid transfection and chemical and physicalmethods such as electroporation. A number of transfection techniques arewell known in the art and are disclosed herein. See, e.g., Graham etal., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, supra; Davis et al., 1986, Basic Methods in MolecularBiology, Elsevier; Chu et al, 1981, Gene 13:197.

The term “SPR” or “surface plasmon resonance”, as used herein, refers toand includes an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Example 5 and Jonsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “fluorescence-activated cell sorting” or “FACS”, as usedherein, refers to a specialized type of flow cytometry. It provides amethod for sorting a heterogeneous mixture of biological cells into twoor more containers, one cell at a time, based upon the specific lightscattering and fluorescent characteristics of each cell (FlowMetric.“Sorting Out Fluorescence Activated Cell Sorting”. Retrieved2017-11-09.). Instruments for carrying out FACS are known to those ofskill in the art and are commercially available to the public. Examplesof such instruments include FACS Star Plus, FACScan and FACSortinstruments from Becton Dickinson (Foster City, Calif.) Epics C fromCoulter Epics Division (Hialeah, Fla.) and MoFlo from Cytomation(Colorado Springs, Colo.).

The term “subject” includes any human or nonhuman animal, preferablyhumans.

The term “condition associated with PD-1” or “condition related toPD-1”, as used herein, refers to any condition that is caused by,exacerbated by, or otherwise linked to increased or decreased expressionor activities of PD-1 (e.g. a human PD-1).

The term “cancer”, as used herein, refers to any or a tumor or amalignant cell growth, proliferation or metastasis-mediated, solidtumors and non-solid tumors such as leukemia and initiate a medicalcondition.

The term “treatment”, “treating” or “treated”, as used herein in thecontext of treating a condition, pertains generally to treatment andtherapy, whether of a human or an animal, in which some desiredtherapeutic effect is achieved, for example, the inhibition of theprogress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.For cancer, “treating” may refer to dampen or slow the tumor ormalignant cell growth, proliferation, or metastasis, or some combinationthereof. For tumors, “treatment” includes removal of all or part of thetumor, inhibiting or slowing tumor growth and metastasis, preventing ordelaying the development of a tumor, or some combination thereof.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen. Specifically, the “therapeutically-effective amount,”refers to an antibody or antigen-binding portion thereof in an amount orconcentration effective to treat the human PD-1-related diseases orconditions.

The present invention in a “host cell”, as used herein, refers to a cellwith the introduction of exogenous polynucleotides.

The term “therapeutically effective amount” or “effective amount”, asused herein, refers to a drug in an amount or concentration effective totreat the human PD-1-related diseases or conditions.

The term “pharmaceutically acceptable”, as used herein, means that thevehicle, diluent, excipient and/or salts thereof, are chemically and/orphysically is compatible with other ingredients in the formulation, andthe physiologically compatible with the recipient.

As used herein, the term “a pharmaceutically acceptable carrier and/orexcipient” refers to a carrier and/or excipient pharmacologically and/orphysiologically compatible with a subject and an active agent, which iswell known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro A R, 19th ed. Pennsylvania: Mack Publishing Company,1995), and includes, but is not limited to pH adjuster, surfactant,adjuvant and ionic strength enhancer. For example, the pH adjusterincludes, but is not limited to, phosphate buffer; the surfactantincludes, but is not limited to, cationic, anionic, or non-ionicsurfactant, e.g., Tween-80; the ionic strength enhancer includes, but isnot limited to, sodium chloride.

As used herein, the term “adjuvant” refers to a non-specificimmunopotentiator, which can enhance immune response to an antigen orchange the type of immune response in an organism when it is deliveredtogether with the antigen to the organism or is delivered to theorganism in advance. There are a variety of adjuvants, including, butnot limited to, aluminium adjuvants (for example, aluminum hydroxide),Freund's adjuvants (for example, Freund's complete adjuvant and Freund'sincomplete adjuvant), coryne bacterium parvum, lipopolysaccharide,cytokines, and the like. Freund's adjuvant is the most commonly usedadjuvant in animal experiments now. Aluminum hydroxide adjuvant is morecommonly used in clinical trials.

PD-1 Binding Molecules

In some aspects, the invention comprises PD-1 binding molecule.

The PD-1 binding molecule, in a general sense, may include any moleculethat specifically binds to PD-1. In some circumstances, “PD-1 bindingmolecule” may include “PD-1 antagonist”. “PD-1 antagonist” refers to anychemical compound or biological molecule that blocks the binding ofPD-L1 to PD-1 expressed on an immune cell (T cell, B cell or NKT cell)and preferably also blocks binding of PD-L2 to the immune-cell expressedPD-1. The PD-1 binding molecule or PD-1 antagonist may be a polypeptideor a protein, for example, an antibody, more particularly, an anti-PD-1antibody.

The antibody includes, but not limited to, a chimeric antibody, ahumanized antibody, or a single-domain antibody. In a specificembodiment, the PD-1 binding molecule is a single-domain antibody, whichgenerally refers to an antibody consisting of a single monomericvariable antibody domain. Like a whole antibody, a single-domainantibody is able to bind selectively to a specific antigen.

More specifically, the PD-1 binding molecule is a single-domain heavychain antibody, which is interchangeably used with the terms “VHH”, “VHHantibody”, “VHH domain”, “VHH antibody fragment”, “V_(HH)” or“Nanobody,” et al. V molecules derived from Camelidae antibodies areamong the smallest intact antigen-binding domains known (approximately15 kDa, or 10 times smaller than a conventional IgG) and hence are wellsuited towards delivery to dense tissues and for accessing the limitedspace between macromolecules.

The single-domain antibody of the invention disclosed herein may be madeby the skilled artisan according to methods known in the art or anyfuture method. For example, VHHs may be obtained using methods known inthe art such as by immunizing a camel and obtaining hybridoma'stherefrom, or by cloning a library of VHHs of the invention usingmolecular biology techniques known in the art and subsequent selectionby using phage display.

For instance, a single-domain antibody can be obtained by immunizationof llamas or alpacas with the desired antigen and subsequent isolationof the mRNA coding for heavy-chain antibodies. By reverse transcriptionand polymerase chain reaction, a gene library of single-domainantibodies containing several million clones is produced. Screeningtechniques like phage display and ribosome display help to identify theclones binding the antigen. One technique is phage display in which alibrary of (e.g., human) antibodies is synthesized on phages, thelibrary is screened with the antigen of interest or an antibody-bindingportion thereof, and the phage that binds the antigen is isolated, fromwhich one may obtain the immunoreactive fragments. Methods for preparingand screening such libraries are well known in the art and kits forgenerating phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Therealso are other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., Barbas et al., Proc.Natl. Acad. Sci. USA 88:7978-7982 (1991)).

When the most potent clones have been identified, their DNA sequence isoptimized, for example, by affinity maturation or humanization.Humanization may prevent immunological reactions of the human organismagainst the antibody.

Accordingly, the single-domain antibodies can be obtained (1) byisolating the VHH domain of a naturally occurring heavy chain antibody;(2) by expression of a nucleotide sequence encoding a naturallyoccurring VHH domain; (3) by “humanization” (as described below) of anaturally occurring VHH domain or by expression of a nucleic acidencoding a such humanized VHH domain; (4) by “camelization” of anaturally occurring VH domain from any animal species, in particular aspecies of mammal, such as from a human being, or by expression of anucleic acid encoding such a camelized VH domain; (5) by “camelisation”of a “domain antibody” or “Dab” as described by Ward et al (supra), orby expression of a nucleic acid encoding such a camelized VH domain; (6)using synthetic or semi-synthetic techniques for preparing proteins,polypeptides or other amino acid sequences; (7) by preparing a nucleicacid encoding a VHH using techniques for nucleic acid synthesis,followed by expression of the nucleic acid thus obtained; and/or (8) byany combination of the foregoing. Suitable methods and techniques forperforming the foregoing will be clear to the skilled person based onthe disclosure herein and for example include the methods and techniquesdescribed in more detail hereinbelow.

Single-domain antibodies are usually generated by PCR cloning ofvariable domain repertoire from blood, lymph node, or spleen cDNAobtained from immunized animals into a phage display vector.Antigen-specific single-domain antibodies are commonly selected bypanning phase libraries on immobilized antigen, e.g., antigen coatedonto the plastic surface of a test tube, biotinylated antigensimmobilized on Streptavidin beads, or membrane proteins expressed on thesurface of cells. The affinity of adAbs can often been improved bymimicking this strategy in vitro, for instance, by site directedmutagenesis of the CDR regions and further rounds of panning onimmobilized antigen under conditions of increased stringency (highertemperature, high or low salt concentration, high or low pH, and lowantigen concentrations) (Wesolowski et al., Single domain antibodies:promising experimental and therapeutic tools in infection and immunity.Med Microbiol Immunol (2009) 198: 157-174).

Methods for preparing a VHH specifically binding to an antigen orepitope was described in references, for example: R. van der Linden etal., Journal of Immunological Methods, 240(2000) 185-195; Li et al., JBiol Chem., 287(2012)13713-13721; Deffar et al., African Journal ofBiotechnology Vol. 8(12), pp. 2645, 17 Jun. 2009 and WO94/04678.

In some embodiments, the VHH in the PD-1 binding molecule is fused to anFc-domain of an antibody, for example, Fc-domain of IgG (e.g., IgG4 orIgG1). In a specific embodiment, the Fc-domain is an Fc-domain of IgG4.By fusing VHH to a Fc domain, it may be more efficient to recruiteffector functions, such as ADCC and CDC. Also, the fusion of VHH to Fcdomain may help the PD-1 binding molecule to form a dimer, and may alsohelp the extension of the half life of PD-1 binding molecule in vivo.

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC”, asused herein, refers to a form of cytotoxicity in which secreted Ig boundonto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.Natural Killer (NK) cells, neutrophils, and macrophages) enable thesecytotoxic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently kill the target cell with cytotoxins. Theantibodies “arm” the cytotoxic cells and are absolutely required forsuch killing. The primary cells for mediating ADCC, NK cells, expressFcyRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. Nos. 5,500,362 or 5,821,337 may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998).

The term “complement dependent cytotoxicity” or “CDC” refers to thelysis of a target cell in the presence of complement. Activation of theclassical complement pathway is initiated by the binding of the firstcomponent of the complement system (C1q) to antibodies (of theappropriate subclass) which are bound to their cognate antigen. Toassess complement activation, a CDC assay, e.g. as described inGazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may beperformed.

For convenience of description, the PD-1 binding molecule is describedas anti-PD-1 antibody in the following sections.

Anti-PD-1 Antibodies with Certain Properties

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. In someembodiments, the antibodies have one or more of the followingproperties:

(a) bind human PD-1 with a K_(D) of 1×10⁻⁷ M or less;

(b) inhibit or blocks binding of PD-L1 or PD-L2 to PD-1;

(c) induce production of IFN-γ in CD4+T cells;

(d) do not substantially bind to human CD28, CTLA-4, ICOS and/or BTL;

(e) have no cross-reactivity with human PD-1, but have cross-reactivitywith mouse PD-1; and

(f) are stable at least 60° C.

The antibody of the invention binds to cell surface PD-1 with highaffinity. The binding of an antibody of the invention to PD-1 can beassessed using one or more techniques well established in the art, forinstance, ELISA. The binding specificity of an antibody of the inventioncan also be determined by monitoring binding of the antibody to cellsexpressing a PD-1 protein, e.g., flow cytometry. For example, anantibody can be tested by a flow cytometry assay in which the antibodyis reacted with a cell line that expresses human PD-1, such as CHO cellsthat have been transfected to express PD-1 on their cell surface.Additionally or alternatively, the binding of the antibody, includingthe binding kinetics (e.g., Kd value) can be tested in BIAcore bindingassays. Still other suitable binding assays include ELISA assays, forexample using a recombinant PD-1 protein. For instance, an antibody ofthe invention binds to a cell surface (e.g., human PD-1) protein with aK_(D) of 1×10⁻⁷ M or less, 5×10⁻⁸ M or less, 2×10⁻⁸ M or less, 5×10⁻⁹ Mor less, 4×10⁻⁹ M or less, 3×10⁻⁹ M or less, 2×10⁻⁹ M or less, 1×10⁻⁹ Mor less, 5×10¹⁰ M or less, 1×10⁻¹⁰ M or less.

In some embodiments, the antibodies of the invention bind to cynomolgusor monkey PD-1 at an EC₅₀ of no more than or about 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM, as measured by FACS.

In some embodiments, the antibodies of the invention inhibit the bindingof human PD-1 to its ligand at an IC₅₀ of 0.2 nM-100 nM (e.g. 0.2 nM-50nM, 0.2 nM-30 nM, 0.2 nM-20 nM, 0.2 nM-10 nM, or 1 nM-10 nM), asmeasured in a competition assay, for example by ELISA.

The anti-PD-1 antibodies of the invention are specific for PD-1. In someembodiments, the antibodies and antigen-binding fragments thereof do notbind to CD28, CTLA-4, ICOS and/or BTL. For example, the binding affinitywith CD28, CTLA-4, ICOS and/or BTL is less than 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1% of the binding affinity with PD-1.

In some embodiments, the antibodies of the invention block binding ofhuman PD-1 to its ligand and thereby providing biological activityincluding, for example, inducing cytokine production from the activatedT cells (such as CD4+ T cells and CD8+ T cells), inducing proliferationof activated T cells (such as CD4+ T cells and CD8+ T cells), andreversing T reg's suppressive function. Exemplary cytokines include IL-2and IFNγ. The term “IL-2” refers to interleukin 2, a type of cytokinesignaling molecule in the immune system that regulates the activities ofwhite blood cells (e.g. leukocytes). The term “Interferon gamma (IFNγ)”is a cytokine that is produced by natural killer (NK), NK T cells, CD4+and CD8+T cells, which is a critical activator of macrophages andinducer of major histocompatibility complex (MEC) molecule expression.The cytokine production can be determined using methods known in theart, for example, by ELISA. Methods can also be used to detectproliferation of T cells, including [3H] thymidine incorporation assay.

In some embodiments, the antibodies of the invention have nocross-reactivity with human PD-1, but have cross-reactivity with mousePD-1. Most of monoclonal antibodies against PD-1 currently tested inclinical trials are only against to human PD-1 which limits preclinicalin vivo assay and diminished efficacy owing to the immunogenicity of themouse-derived protein sequences. Humanized antibody withcross-reactivity to mouse PD-1 overcome these shortages and showed moretolerability and higher efficiency in vivo.

Anti-PD-1 Antibodies Comprising CDRs with Sequence Identity to SpecificSequences

In some embodiments, the anti-PD-1 antibody of the invention comprisesat least one immunoglobulin single variable domain (for example, VHH),wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1comprises an amino acid sequence which is at least 90% identical to SEQID NO: 1, CDR2 comprises an amino acid sequence which is at least 90%identical to SEQ ID NO: 2, and CDR3 comprises an amino acid sequencewhich is at least 80% identical to SEQ ID NO: 3.

The assignment of amino acids to each CDR may be in accordance with oneof the numbering schemes provided by Kabat et al. (1991) Sequences ofProteins of Immunological Interest (5^(th) Ed.), US Dept. of Health andHuman Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al.,1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum etal., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of TherapeuticAntibodies, 3^(rd) Ed., Wily-VCH Verlag GmbH and Co. unless otherwisenoted.

Variable regions and CDRs in an antibody sequence can be identifiedaccording to general rules that have been developed in the art (as setout above, such as, for example, the Kabat numbering system) or byaligning the sequences against a database of known variable regions.Methods for identifying these regions are described in Kontermann andDubel, eds., Antibody Engineering, Springer, New York, N.Y., 2001 andDinarello et al., Current Protocols in Immunology, John Wiley and SonsInc., Hoboken, N.J., 2000. Exemplary databases of antibody sequences aredescribed in, and can be accessed through, the “Abysis” website atwww.bioinf.org.uk/abs (maintained by A.C. Martin in the Department ofBiochemistry & Molecular Biology University College London, London,England) and the VBASE2 website at www.vbase2.org, as described inRetter et al., Nucl. Acids Res., 33 (Database issue): D671 -D674 (2005).Preferably sequences are analyzed using the Abysis database, whichintegrates sequence data from Kabat, IMGT and the Protein Data Bank(PDB) with structural data from the PDB. See Dr. Andrew C. R. Martin'sbook chapter Protein Sequence and Structure Analysis of AntibodyVariable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S.and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available on the website bioinforg.uk/abs). TheAbysis database website further includes general rules that have beendeveloped for identifying CDRs which can be used in accordance with theteachings herein. Unless otherwise indicated, all CDRs set forth hereinare derived according to the Abysis database website as per Kabat.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percentage of identitybetween two amino acid sequences can be determined by the algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al, (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs {e.g.,XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

In other embodiments, the CDR amino acid sequences can be at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to therespective sequences set forth above.

Anti-PD-1 Antibodies Comprising CDRs with Amino Acid Addition, Deletionor Substitution

In some embodiments, the anti-PD-1 antibody of the invention comprisesat least one immunoglobulin single variable domain (for example, VHH),wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 differsin amino acid sequence from SEQ ID NO: 1 by an amino acid addition,deletion or substitution of not more than 2 amino acids; CDR2 differs inamino acid sequence from SEQ ID NO: 2 by an amino acid addition,deletion or substitution of not more than 2 amino acids; and/or CDR3differs in amino acid sequence from SEQ ID NO: 3 by an amino acidaddition, deletion or substitution of not more than 2 amino acids. Forexamples, the CDR1, CDR2 and CDR3 differs from amino acid sequences setforth in SEQ ID NOs: 1-3 by an amino acid addition, deletion orsubstitution of only one amino acid, respectively.

In some embodiments, the anti-PD-1 antibody of the invention comprisesat least one immunoglobulin single variable domain (for example, VHH),wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1, CDR2and CDR3 are selected from the group comprising:

(a) CDR1 which is represented by DSIX₁SX₂VNMG, wherein X₁=D or Q, andX₂=M or L;

(b) CDR2 which is represented by LIAX₃YITHYADFVKG, wherein X₃=N, T, Y, Ror W;

(c) CDR3 which is represented by RX₄IX₅X₆DY, wherein X₄=N or S, X₅=I, Ror Y, and X₆=V or E.

Preferably, the CDRs of the isolated antibody or the antigen-bindingportion thereof contain a conservative substitution of not more than 2amino acids, or not more than 1 amino acid. The term “conservativesubstitution”, as used herein, refers to amino acid substitutions whichwould not disadvantageously affect or change the essential properties ofa protein/polypeptide comprising the amino acid sequence. For example, aconservative substitution may be introduced by standard techniques knownin the art such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include substitutionswherein an amino acid residue is substituted with another amino acidresidue having a similar side chain, for example, a residue physicallyor functionally similar (such as, having similar size, shape, charge,chemical property including the capability of forming covalent bond orhydrogen bond, etc.) to the corresponding amino acid residue. Thefamilies of amino acid residues having similar side chains have beendefined in the art. These families include amino acids having alkalineside chains (for example, lysine, arginine and histidine), amino acidshaving acidic side chains (for example, aspartic acid and glutamicacid), amino acids having uncharged polar side chains (for example,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), amino acids having nonpolar side chains (for example,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), amino acids having β-branched side chains (such asthreonine, valine, isoleucine) and amino acids having aromatic sidechains (for example, tyrosine, phenylalanine, tryptophan, histidine).Therefore, a corresponding amino acid residue is preferably substitutedwith another amino acid residue from the same side-chain family. Methodsfor identifying amino acid conservative substitutions are well known inthe art (see, for example, Brummell et al., Biochem. 32: 1180-1187(1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burkset al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which areincorporated herein by reference).

Anti-PD-1 Antibodies Comprising CDRs

In some embodiments, at least one immunoglobulin single variable domain(for example, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, andwherein CDR1, CDR2 and CDR3 are selected from the group comprising:

(a) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 1, CDR2 comprising or consisting of an amino acid sequence asshown in SEQ ID NO: 2, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 3;

(b) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 4, CDR2 comprising or consisting of an amino acid sequence asshown in SEQ ID NO: 5, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 6;

(c) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 7, CDR2 comprising or consisting of an amino acid sequence asshown in SEQ ID NO: 8, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 9;

(d) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 10, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 11, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 12;

(e) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 13, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 14, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 15;

(f) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 16, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 17, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 18;

(g) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 19, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 20, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 21;

(h) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 22, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 23, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 24;

(i) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 25, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 26, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 27;

(j) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 28, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 29, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 30;

(k) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 31, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 32, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 33; and

(l) CDR1 comprising or consisting of an amino acid sequence as shown inSEQ ID NO: 34, CDR2 comprising or consisting of an amino acid sequenceas shown in SEQ ID NO: 35, and CDR3 comprising or consisting of an aminoacid sequence as shown in SEQ ID NO: 36.

In some embodiments, at least one immunoglobulin single variable domain(for example, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, andwherein CDR1, CDR2 and CDR3 are selected from the group comprising:

(a) CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 withan amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 3;

(b) CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, CDR2 withan amino acid sequence as shown in SEQ ID NO: 5, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 6;

(c) CDR1 with an amino acid sequence as shown in SEQ ID NO: 7, CDR2 withan amino acid sequence as shown in SEQ ID NO: 8, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 9;

(d) CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, CDR2with an amino acid sequence as shown in SEQ ID NO: 11, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 12;

(e) CDR1 with an amino acid sequence as shown in SEQ ID NO: 13, CDR2with an amino acid sequence as shown in SEQ ID NO: 14, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 15;

(f) CDR1 with an amino acid sequence as shown in SEQ ID NO: 16, CDR2with an amino acid sequence as shown in SEQ ID NO: 17, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 18;

(g) CDR1 with an amino acid sequence as shown in SEQ ID NO: 19, CDR2with an amino acid sequence as shown in SEQ ID NO: 20, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 21;

(h) CDR1 with an amino acid sequence as shown in SEQ ID NO: 22, CDR2with an amino acid sequence as shown in SEQ ID NO: 23, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 24;

(i) CDR1 with an amino acid sequence as shown in SEQ ID NO: 25, CDR2with an amino acid sequence as shown in SEQ ID NO: 26, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 27;

(j) CDR1 with an amino acid sequence as shown in SEQ ID NO: 28, CDR2with an amino acid sequence as shown in SEQ ID NO: 29, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 30;

(k) CDR1 with an amino acid sequence as shown in SEQ ID NO: 31, CDR2with an amino acid sequence as shown in SEQ ID NO: 32, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 33; and

(l) CDR1 with an amino acid sequence as shown in SEQ ID NO: 34, CDR2with an amino acid sequence as shown in SEQ ID NO: 35, and CDR3 with anamino acid sequence as shown in SEQ ID NO: 36.

Anti-PD-1 Antibodies Defined via the Sequence of VHH

In some embodiments, the anti-PD-1 antibodies comprises at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises or consists of:

-   (A) the amino acid sequence shown in any of SEQ ID NOs: 37-49;-   (B) an amino acid sequence which is at least 85%, at least 90%, or    at least 95% identical to any of SEQ ID NOs: 37-49; or-   (C) an amino acid sequence with addition, deletion and/or    substitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9    or 10) amino acids compared with any of SEQ ID NOs: 37-49.

In other embodiments, the amino acid sequences of VHH can be at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the respective sequences set forth above. As anillustrative example, the antibody may comprise a VHH with at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO: 37.

In some further embodiments, the anti-PD-1 antibodies may containconservative substitution or modification of amino acids in the variableregions of the heavy chain and/or light chain. It is understood in theart that certain conservative sequence modification can be made which donot remove antigen binding. See, e.g., Brummell et al. (1993) Biochem32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov etal. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J.Immunol. 149:1605-12; Kelley and 0′ Connell (1993) Biochem. 32:6862-35;Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000)Clin. Can. Res. 6:2835-43.

As described above, the term “conservative substitution”, as usedherein, refers to amino acid substitutions which would notdisadvantageously affect or change the essential properties of aprotein/polypeptide comprising the amino acid sequence. For example, aconservative substitution may be introduced by standard techniques knownin the art such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include substitutionswherein an amino acid residue is substituted with another amino acidresidue having a similar side chain, for example, a residue physicallyor functionally similar (such as, having similar size, shape, charge,chemical property including the capability of forming covalent bond orhydrogen bond, etc.) to the corresponding amino acid residue. Thefamilies of amino acid residues having similar side chains have beendefined in the art. These families include amino acids having alkalineside chains (for example, lysine, arginine and histidine), amino acidshaving acidic side chains (for example, aspartic acid and glutamicacid), amino acids having uncharged polar side chains (for example,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), amino acids having nonpolar side chains (for example,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), amino acids having β-branched side chains (such asthreonine, valine, isoleucine) and amino acids having aromatic sidechains (for example, tyrosine, phenylalanine, tryptophan, histidine).Therefore, a corresponding amino acid residue is preferably substitutedwith another amino acid residue from the same side-chain family. Methodsfor identifying amino acid conservative substitutions are well known inthe art (see, for example, Brummell et al., Biochem. 32: 1180-1187(1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burkset al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which areincorporated herein by reference).

Binning and Epitope Mapping

It will further be appreciated the disclosed antibodies will associatewith, or bind to, discrete epitopes or immunogenic determinantspresented by the selected target or fragment thereof. In someembodiments, epitope or immunogenic determinants include chemicallyactive surface groupings of molecules such as amino acids, sugar sidechains, phosphoryl groups, or sulfonyl groups. In some embodiments,epitopes may have specific three-dimensional structural characteristics,and/or specific charge characteristics. Thus, as used herein the term“epitope” includes any protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor or otherwise interacting with amolecule. In some embodiments, an antibody is said to specifically bind(or immune-specifically bind or react) an antigen when it preferentiallyrecognizes its target antigen in a complex mixture of proteins and/ormacromolecules. In some embodiments, an antibody is said to specificallybind an antigen when the equilibrium dissociation constant (K_(D)) isless than or equal to 10⁻⁶M or less than or equal to 10⁻⁷M, morepreferably when the e K_(D) is less than or equal to 10⁻⁸M, and evenmore preferably when the K_(D) is less than or equal to 10⁻⁹M.

Epitopes formed from contiguous amino acids (sometimes referred to as“linear” or “continuous” epitopes) are typically retained upon proteindenaturing, whereas epitopes formed by tertiary folding are typicallylost upon protein denaturing. In any event an antibody epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

In this respect, it will be appreciated that, in some embodiments, anepitope may be associated with, or reside in, one or more regions,domains or motifs of, for example, the PD-1 protein. Similarly, theart-recognized term “motif” will be used in accordance with its commonmeaning and shall generally refer to a short, conserved region of aprotein that is typically ten to twenty contiguous amino acid residues.

In any event once a desired epitope on an antigen is determined, it ispossible to generate antibodies to that epitope, e.g., by immunizingwith a peptide comprising the epitope using techniques described in thepresent invention. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes located in specific domains or motifs. Fromthis information, it is then possible to competitively screen antibodiesfor binding to the same epitope. An approach to achieve this is toconduct competition studies to find antibodies that competitively bindwith one another, i.e. the antibodies compete for binding to theantigen. A high throughput process for binning antibodies based upontheir cross-competition is described in WO 03/48731. Other methods ofbinning or domain level or epitope mapping comprising antibodycompetition or antigen fragment expression on yeast are well known inthe art.

As used herein, the term “binning” refers to methods used to group orclassify antibodies based on their antigen binding characteristics andcompetition. While the techniques are useful for defining andcategorizing the antibodies of the instant invention, the bins do notalways directly correlate with epitopes and such initial determinationsof epitope binding may be further refined and confirmed by otherart-recognized methodology in the art and as described herein. However,it will be appreciated that empirical assignment of the antibodies toindividual bins provides information that may be indicative of thetherapeutic potential of the disclosed antibodies.

More specifically, one can determine whether a selected referenceantibody (or fragment thereof) binds to the same epitope or crosscompetes for binding with a second test antibody (i.e., is in the samebin) by using methods known in the art and set forth in the Examplesherein.

Other compatible epitope mapping techniques include alanine scanningmutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63)(herein specifically incorporated by reference in its entirety), orpeptide cleavage analysis. In addition, methods such as epitopeexcision, epitope extraction and chemical modification of antigens canbe employed (Tomer (2000) Protein Science 9: 487-496) (hereinspecifically incorporated by reference in its entirety).

Nucleic Acid Molecules Encoding Antibodies of the Invention

In some aspects, the invention is directed to an isolated nucleic acidmolecule, comprising a nucleic acid sequence encoding VHHs as disclosedherein.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), a nucleic acid encodingsuch antibodies can be recovered from the gene library.

Exemplary nucleic acids molecules of the invention are those set forthSEQ ID Nos: 50-62, respectively. In some embodiments, the nucleic acidsshare an at least 80% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ IDNOs: 17-20, respectively. In some embodiments, the percentage ofidentity is derived from the degeneracy of the genetic code, and theencoded protein sequences remain unchanged.

The nucleic acid molecules that encodes the anti-PD-1 antibodies can beinserted into a vector for further cloning (amplification of the DNA) orfor expression, using recombinant techniques known in the art. Inanother embodiment, the antibody may be produced by homologousrecombination known in the art. DNA encoding the monoclonal antibody isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy chain of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter(e.g. SV40, CMV, EF-1α), and a transcription termination sequence. Theselectable marker gene facilitates selection of host cells into whichthe vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216;4,634,665 and 5,179,017). For example, typically the selectable markergene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Selectable marker genes may include the dihydrofolate reductase (DHFR)gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

In some embodiments, the vector system includes mammalian, bacterial,yeast systems, etc, and comprises plasmids such as, but not limited to,pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP,pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO,Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2etc, and other laboratorial and commercially available vectors. Suitablevectors may include, plasmid, or viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses). Inone embodiment of the invention, the vector may be pET, for instance,pETbac containing genes of hexa-histidine- and c-Myc-tag.

Vectors comprising the nucleic acid sequence encoding the PD-1 bindingmolecule can be introduced to a host cell for cloning or geneexpression. Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis, Pseudomonas such as P.aeruginosa, and Streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-PD-1antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Other suitable host cells for the expression of the anti-PD-1 antibodiesprovided here are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruiffly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts.

Host cells are transformed with the above-described expression orcloning vectors for anti-PD-1 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the anti-PD-1 antibodies provided hereinmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody prepared from the cells can be purified using, for example,hydroxylapatite chromatography, gel electrophoresis, dialysis,DEAE-cellulose ion exchange chromatography, ammonium sulfateprecipitation, salting out, and affinity chromatography, with affinitychromatography being the preferred purification technique.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Compositions

In some aspects, the invention is directed to a pharmaceuticalcomposition comprising at least one PD-1 binding molecule as disclosedherein and a pharmaceutically acceptable carrier.

Components of the Compositions

The pharmaceutical composition may optionally contain one or moreadditional pharmaceutically active ingredients, such as another antibodyor a drug. The pharmaceutical compositions of the invention also can beadministered in a combination therapy with, for example, anotherimmune-stimulatory agent, anti-cancer agent, an antiviral agent, or avaccine, such that the anti-PD-1 antibody enhances the immune responseagainst the vaccine. A pharmaceutically acceptable carrier can include,for example, a pharmaceutically acceptable liquid, gel or solidcarriers, an aqueous medium, a non-aqueous medium, an anti-microbialagent, isotonic agents, buffers, antioxidants, anesthetics,suspending/dispersing agent, a chelating agent, a diluent, adjuvant,excipient or a nontoxic auxiliary substance, other known in the artvarious combinations of components or more.

Suitable components may include, for example, antioxidants, fillers,binders, disintegrating agents, buffers, preservatives, lubricants,flavorings, thickening agents, coloring agents, emulsifiers orstabilizers such as sugars and cyclodextrin. Suitable anti-oxidants mayinclude, for example, methionine, ascorbic acid, EDTA, sodiumthiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole,butylated hydroxy toluene and/or propylgalacte. As disclosed in thepresent invention, in a solvent containing an antibody or anantigen-binding fragment of the present invention discloses compositionsinclude one or more anti-oxidants such as methionine, reducing antibodyor antigen binding fragment thereof may be oxidized. The oxidationreduction may prevent or reduce a decrease in binding affinity, therebyenhancing antibody stability and extended shelf life. Thus, in someembodiments, the present invention provides a composition comprising oneor more antibodies or antigen binding fragment thereof and one or moreanti-oxidants such as methionine. The present invention further providesa variety of methods, wherein an antibody or antigen binding fragmentthereof is mixed with one or more anti-oxidants, such as methionine, sothat the antibody or antigen binding fragment thereof can be preventedfrom oxidation, to extend their shelf life and/or increased activity.

To further illustrate, pharmaceutical acceptable carriers may include,for example, aqueous vehicles such as sodium chloride injection,Ringer's injection, isotonic dextrose injection, sterile waterinjection, or dextrose and lactated Ringer's injection, nonaqueousvehicles such as fixed oils of vegetable origin, cottonseed oil, cornoil, sesame oil, or peanut oil, antimicrobial agents at bacteriostaticor fungistatic concentrations, isotonic agents such as sodium chlorideor dextrose, buffers such as phosphate or citrate buffers, antioxidantssuch as sodium bisulfate, local anesthetics such as procainehydrochloride, suspending and dispersing agents such as sodiumcarboxymethylcelluose, hydroxypropyl methylcellulose, orpolyvinylpyrrolidone, emulsifying agents such as Polysorbate 80(TWEEN-80), sequestering or chelating agents such as EDTA(ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraaceticacid), ethyl alcohol, polyethylene glycol, propylene glycol, sodiumhydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobialagents utilized as carriers may be added to pharmaceutical compositionsin multiple-dose containers that include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Suitable excipients may include, for example, water, saline, dextrose,glycerol, or ethanol. Suitable non-toxic auxiliary substances mayinclude, for example, wetting or emulsifying agents, pH bufferingagents, stabilizers, solubility enhancers, or agents such as sodiumacetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

Administration, Formulation and Dosage

The pharmaceutical composition of the invention may be administered invivo, to a subject in need thereof, by various routes, including, butnot limited to, oral, intravenous, intra-arterial, subcutaneous,parenteral, intranasal, intramuscular, intracranial, intracardiac,intraventricular, intratracheal, buccal, rectal, intraperitoneal,intradermal, topical, transdermal, and intrathecal, or otherwise byimplantation or inhalation. The subject compositions may be formulatedinto preparations in solid, semi-solid, liquid, or gaseous forms;including, but not limited to, tablets, capsules, powders, granules,ointments, solutions, suppositories, enemas, injections, inhalants, andaerosols. The appropriate formulation and route of administration may beselected according to the intended application and therapeutic regimen.

Suitable formulations for enteral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Similarly,the particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory, as well as empirical considerations such as pharmacokinetics(e.g., half-life, clearance rate, etc.).

Frequency of administration may be determined and adjusted over thecourse of therapy, and is based on reducing the number of proliferativeor tumorigenic cells, maintaining the reduction of such neoplasticcells, reducing the proliferation of neoplastic cells, or delaying thedevelopment of metastasis. In some embodiments, the dosage administeredmay be adjusted or attenuated to manage potential side effects and/ortoxicity. Alternatively, sustained continuous release formulations of asubject therapeutic composition may be appropriate.

It will be appreciated by one of skill in the art that appropriatedosages can vary from patient to patient. Determining the optimal dosagewill generally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action that achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

In general, the PD-1 binding molecules may be administered in variousranges. In some embodiments, the PD-1 binding molecules as providedherein may be administered at a therapeutically effective dosage ofabout 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg,about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100mg/kg) . In certain of these embodiments, the antibody is administeredat a dosage of about 50 mg/kg or less, and in certain of theseembodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg orless, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments,the administration dosage may change over the course of treatment. Forexample, in certain embodiments the initial administration dosage may behigher than subsequent administration dosages. In certain embodiments,the administration dosage may vary over the course of treatmentdepending on the reaction of the subject.

In any event, the antibody or the antigen binding portion thereof of theinvention is preferably administered as needed to subjects in needthereof. Determination of the frequency of administration may be made bypersons skilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like.

In certain preferred embodiments, the course of treatment involving theantibody or the antigen-binding portion thereof of the instant inventionwill comprise multiple doses of the selected drug product over a periodof weeks or months. More specifically, the antibody or theantigen-binding portion thereof of the instant invention may beadministered once every day, every two days, every four days, everyweek, every ten days, every two weeks, every three weeks, every month,every six weeks, every two months, every ten weeks or every threemonths. In this regard, it will be appreciated that the dosages may bealtered or the interval may be adjusted based on patient response andclinical practices.

Dosages and regimens may also be determined empirically for thedisclosed therapeutic compositions in individuals who have been givenone or more administration(s). For example, individuals may be givenincremental dosages of a therapeutic composition produced as describedherein. In selected embodiments, the dosage may be gradually increasedor reduced or attenuated based respectively on empirically determined orobserved side effects or toxicity. To assess efficacy of the selectedcomposition, a marker of the specific disease, disorder or condition canbe followed as described previously. For cancer, these include directmeasurements of tumor size via palpation or visual observation, indirectmeasurement of tumor size by x-ray or other imaging techniques; animprovement as assessed by direct tumor biopsy and microscopicexamination of the tumor sample; the measurement of an indirect tumormarker (e.g., PSA for prostate cancer) or a tumorigenic antigenidentified according to the methods described herein, a decrease in painor paralysis; improved speech, vision, breathing or other disabilityassociated with the tumor; increased appetite; or an increase in qualityof life as measured by accepted tests or prolongation of survival. Itwill be apparent to one of skill in the art that the dosage will varydepending on the individual, the type of neoplastic condition, the stageof neoplastic condition, whether the neoplastic condition has begun tometastasize to other location in the individual, and the past andconcurrent treatments being used.

Compatible formulations for parenteral administration (e.g., intravenousinjection) may comprise the PD-1 binding molecules as provided herein inconcentrations of from about 10 μg/ml to about 100 mg/ml. In someembodiments, the concentrations of the PD-1 binding molecule maycomprise 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml,300, μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900μg/ml or 1 mg/ml. In other preferred embodiments, the concentration ofthe PD-1 binding molecule comprise 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml, 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

Applications of the Invention

The PD-1 binding molecules of the present invention have numerous invitro and in vivo utilities. For example, these molecules can beadministered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.The immune response can be augmented, stimulated or up-regulated.

Preferred subjects include human patients in need of enhancement of animmune response. The methods are particularly suitable for treatinghuman patients having a disorder that can be treated by augmenting animmune response (e.g., the T-cell mediated immune response). In aparticular embodiment, the methods are particularly suitable fortreatment of cancer in vivo. To achieve antigen-specific enhancement ofimmunity, the anti-PD-1 antibodies can be administered together with anantigen of interest or the antigen may already be present in the subjectto be treated (e.g., a tumor-bearing or virus-bearing subject). Whenanti-PD-1 antibodies are administered together with another agent, thetwo can be administered in either order or simultaneously.

The invention further provides methods for detecting the presence ofPD-1 antigen in a sample, or measuring the amount of human PD-1 antigen,comprising contacting the sample, and a control sample, with the PD-1binding molecules, under conditions that allow for formation of acomplex between the PD-1 binding molecule and PD-1. The formation of acomplex is then detected, wherein a difference complex formation betweenthe sample compared to the control sample is indicative of the presenceof PD-1 antigen in the sample. Moreover, the PD-1 binding molecules ofthe invention can be used to purify human PD-1 via immunoaffinitypurification.

Treatment of Cancers

Conditions and disorders associated with PD-1 can be an immunity-relateddisease or disorder. In some embodiments, the PD-1 associated conditionsand disorders include tumors and cancers, for example, non-small celllung cancer, small cell lung cancer, renal cell cancer, colorectalcancer, ovarian cancer, breast cancer, pancreatic cancer, gastriccarcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma,head and neck cancer, thyroid cancer, sarcoma, prostate cancer,glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas,myelomas, mycoses fungoids, merkel cell cancer, and other hematologicmalignancies, such as classical Hodgkin lymphoma (CHL), primarymediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-celllymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuselarge B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodalNK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associatedprimary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the centralnervous system (CNS), such as primary CNS lymphoma, spinal axis tumor,brain stem glioma. In certain embodiments, the tumors and cancers aremetastatic, especially metastatic tumors expressing PD-L1. In certainembodiments, the PD-1 associated conditions and disorders includeautoimmune diseases, such as systemic lupus erythematosus (SLE),psoriasis, systemic scleroderma, autoimmune diabetes and the like. Incertain embodiments, the PD-1 associated conditions and disordersinclude infectious disease such as chronic viral infection, for example,viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barrvirus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplexvirus type 2, human papilloma virus, adenovirus, Kaposi West sarcomaassociated herpes virus epidemics, thin ring virus (Torquetenovirus), JCvirus or BK virus.

The antibody or the antigen-binding portion thereof may be used incombination with chemical therapies or radiotherapies.

Combined use with Chemotherapies

The antibody or the antigen-binding portion thereof may be used incombination with an anti-cancer agent, a cytotoxic agent orchemotherapeutic agent.

The term “anti-cancer agent” or “anti-proliferative agent” means anyagent that can be used to treat a cell proliferative disorder such ascancer, and includes, but is not limited to, cytotoxic agents,cytostatic agents, anti-angiogenic agents, debulking agents,chemotherapeutic agents, radiotherapy and radiotherapeutic agents,targeted anti-cancer agents, BRMs, therapeutic antibodies, cancervaccines, cytokines, hormone therapies, radiation therapy andanti-metastatic agents and immunotherapeutic agents. It will beappreciated that, in selected embodiments as discussed above, suchanti-cancer agents may comprise conjugates and may be associated withthe disclosed site-specific antibodies prior to administration. Morespecifically, In some embodiments selected anti-cancer agents will belinked to the unpaired cysteines of the engineered antibodies to provideengineered conjugates as set forth herein. Accordingly, such engineeredconjugates are expressly contemplated as being within the scope of theinstant invention. In other embodiments, the disclosed anti-canceragents will be given in combination with site-specific conjugatescomprising a different therapeutic agent as set forth above.

As used herein the term “cytotoxic agent” means a substance that istoxic to the cells and decreases or inhibits the function of cellsand/or causes destruction of cells. In some embodiments, the substanceis a naturally occurring molecule derived from a living organism.Examples of cytotoxic agents include, but are not limited to, smallmolecule toxins or enzymatically active toxins of bacteria (e.g.,Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcalenterotoxin A), fungal (e.g., α-sarcin, restrictocin), plants (e.g.,abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin,gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordiiproteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin,neomycin, and the tricothecenes) or animals, (e.g., cytotoxic RNases,such as extracellular pancreatic RNases; DNase I, including fragmentsand/or variants thereof).

For the purposes of the instant invention a “chemotherapeutic agent”comprises a chemical compound that non-specifically decreases orinhibits the growth, proliferation, and/or survival of cancer cells(e.g., cytotoxic or cytostatic agents). Such chemical agents are oftendirected to intracellular processes necessary for cell growth ordivision, and are thus particularly effective against cancerous cells,which generally grow and divide rapidly. For example, vincristinedepolymerizes microtubules, and thus inhibits cells from enteringmitosis. In general, chemotherapeutic agents can include any chemicalagent that inhibits, or is designed to inhibit, a cancerous cell or acell likely to become cancerous or generate tumorigenic progeny (e.g.,TIC). Such agents are often administered, and are often most effective,in combination, e.g., in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with thesite-specific constructs of the present invention (either as a componentof a site specific conjugate or in an unconjugated state) include, butare not limited to, alkylating agents, alkyl sulfonates, aziridines,ethylenimines and methylamelamines, acetogenins, a camptothecin,bryostatin, callystatin, CC-1065, cryptophycins, dolastatin,duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin,nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin,bisphosphonates, esperamicin, chromoprotein enediyne antiobioticchromophores, aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib,vemurafenib, crizotinib,sorafenib, ibrutinib, enzalutamide, folic acidanalogues, purine analogs, androgens, anti-adrenals, folic acidreplenisher such as frolinic acid, aceglatone, aldophosphamideglycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil,bisantrene, edatraxate, defofamine, demecolcine, diaziquone,elfornithine, elliptinium acetate, an epothilone, etoglucid, galliumnitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs, vinblastine; platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; difluorometlhylornithine; retinoids; capecitabine; combretastatin;leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR andVEGF-A that reduce cell proliferation and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators, aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,and anti-androgens; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, ribozymes such as a VEGFexpression inhibitor and a HER2 expression inhibitor; vaccines,PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH;Vinorelbine and Esperamicins and pharmaceutically acceptable salts,acids or derivatives of any of the above.

Combined use with Radiotherapies

The present invention also provides for the combination of the antibodyor the antigen-binding portion thereof with radiotherapy (i.e., anymechanism for inducing DNA damage locally within tumor cells such asgamma-irradiation, X-rays, UV-irradiation, microwaves, electronicemissions and the like). Combination therapy using the directed deliveryof radioisotopes to tumor cells is also contemplated, and the disclosedconjugates may be used in connection with a targeted anti-cancer agentor other targeting means. Typically, radiation therapy is administeredin pulses over a period of time from about 1 to about 2 weeks. Theradiation therapy may be administered to subjects having head and neckcancer for about 6 to 7 weeks. Optionally, the radiation therapy may beadministered as a single dose or as multiple, sequential doses.

Diagnosis

The invention provides in vitro and in vivo methods for detecting,diagnosing or monitoring proliferative disorders and methods ofscreening cells from a patient to identify tumor cells includingtumorigenic cells. Such methods include identifying an individual havingcancer for treatment or monitoring progression of a cancer, comprisingcontacting the patient or a sample obtained from a patient (either invivo or in vitro) with an antibody as described herein and detectingpresence or absence, or level of association, of the antibody to boundor free target molecules in the sample. In some embodiments, theantibody will comprise a detectable label or reporter molecule asdescribed herein.

In some embodiments, the association of the antibody with particularcells in the sample can denote that the sample may contain tumorigeniccells, thereby indicating that the individual having cancer may beeffectively treated with an antibody as described herein.

Samples can be analyzed by numerous assays, for example,radioimmunoassays, enzyme immunoassays (e.g. ELISA), competitive-bindingassays, fluorescent immunoassays, immunoblot assays, Western Blotanalysis and flow cytometry assays. Compatible in vivo theragnostic ordiagnostic assays can comprise art recognized imaging or monitoringtechniques, for example, magnetic resonance imaging, computerizedtomography (e.g. CAT scan), positron tomography (e.g., PET scan),radiography, ultrasound, etc., as would be known by those skilled in theart.

Pharmaceutical Packs and Kits

Pharmaceutical packs and kits comprising one or more containers,comprising one or more doses of the antibody or the antigen-bindingportion thereof are also provided. In some embodiments, a unit dosage isprovided wherein the unit dosage contains a predetermined amount of acomposition comprising, for example, the antibody or the antigen-bindingportion thereof, with or without one or more additional agents. Forother embodiments, such a unit dosage is supplied in single-useprefilled syringe for injection. In still other embodiments, thecomposition contained in the unit dosage may comprise saline, sucrose,or the like; a buffer, such as phosphate, or the like; and/or beformulated within a stable and effective pH range. Alternatively, Insome embodiments, the conjugate composition may be provided as alyophilized powder that may be reconstituted upon addition of anappropriate liquid, for example, sterile water or saline solution. Incertain preferred embodiments, the composition comprises one or moresubstances that inhibit protein aggregation, including, but not limitedto, sucrose and arginine. Any label on, or associated with, thecontainer(s) indicates that the enclosed conjugate composition is usedfor treating the neoplastic disease condition of choice.

The present invention also provides kits for producing single-dose ormulti-dose administration units of site-specific conjugates and,optionally, one or more anti-cancer agents. The kit comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic and contain a pharmaceutically effective amountof the disclosed conjugates in a conjugated or unconjugated form. Inother preferred embodiments, the container(s) comprise a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Suchkits will generally contain in a suitable container a pharmaceuticallyacceptable formulation of the engineered conjugate and, optionally, oneor more anti-cancer agents in the same or different containers. The kitsmay also contain other pharmaceutically acceptable formulations, eitherfor diagnosis or combined therapy. For example, in addition to theantibody or the antigen-binding portion thereof of the invention suchkits may contain any one or more of a range of anti-cancer agents suchas chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents;anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents;and/or other anti-cancer agents.

More specifically the kits may have a single container that contains thedisclosed the antibody or the antigen-binding portion thereof, with orwithout additional components, or they may have distinct containers foreach desired agent. Where combined therapeutics are provided forconjugation, a single solution may be pre-mixed, either in a molarequivalent combination, or with one component in excess of the other.Alternatively, the conjugates and any optional anti-cancer agent of thekit may be maintained separately within distinct containers prior toadministration to a patient. The kits may also comprise a second/thirdcontainer means for containing a sterile, pharmaceutically acceptablebuffer or other diluent such as bacteriostatic water for injection(BWFI), phosphate-buffered saline (PBS), Ringer's solution and dextrosesolution.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous or saline solution being particularly preferred.However, the components of the kit may be provided as dried powder(s).When reagents or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

As indicated briefly above the kits may also contain a means by which toadminister the antibody or the antigen-binding portion thereof and anyoptional components to a patient, e.g., one or more needles, I.V. bagsor syringes, or even an eye dropper, pipette, or other such likeapparatus, from which the formulation may be injected or introduced intothe animal or applied to a diseased area of the body. The kits of thepresent invention will also typically include a means for containing thevials, or such like, and other component in close confinement forcommercial sale, such as, e.g., injection or blow-molded plasticcontainers into which the desired vials and other apparatus are placedand retained.

Sequence Listing Summary

Appended to the instant application is a sequence listing comprising anumber of nucleic acid and amino acid sequences. The following Table Aprovides a summary of the included sequences.

TABLE A SEQ ID NO. Description 1 CDR1 of AP17R1-2H2 or AP17R1-2H2-Z1 2CDR2 of AP17R1-2H2 or AP17R1-2H2-Z1 3 CDR3 of AP17R1-2H2 orAP17R1-2H2-Z1 4 CDR1 of AP17R1-2H2-Z1-R1-4B2 5 CDR2 ofAP17R1-2H2-Z1-R1-4B2 6 CDR3 of AP17R1-2H2-Z1-R1-4B2 7 CDR1 ofAP17R1-2H2-Z1-R1-4D8 8 CDR2 of AP17R1-2H2-Z1-R1-4D8 9 CDR3 ofAP17R1-2H2-Z1-R1-4D8 10 CDR1 of AP17R1-2H2-Z1-R1-6E1 11 CDR2 ofAP17R1-2H2-Z1-R1-6E1 12 CDR3 of AP17R1-2H2-Z1-R1-6E1 13 CDR1 ofAP17R1-2H2-Z1-R1-14A1 14 CDR2 of AP17R1-2H2-Z1-R1-14A1 15 CDR3 ofAP17R1-2H2-Z1-R1-14A1 16 CDR1 of AP17R1-2H2-Z1-R1-14F1 17 CDR2 ofAP17R1-2H2-Z1-R1-14F1 18 CDR3 of AP17R1-2H2-Z1-R1-14F1 19 CDR1 ofAP17R1-2H2-Z1-R1-14B3 20 CDR2 of AP17R1-2H2-Z1-R1-14B3 21 CDR3 ofAP17R1-2H2-Z1-R1-14B3 22 CDR1 of AP17R1-2H2-Z1-R1-14F3 23 CDR2 ofAP17R1-2H2-Z1-R1-14F3 24 CDR3 of AP17R1-2H2-Z1-R1-14F3 25 CDR1 ofAP17R1-2H2-Z1-R1-27A2 26 CDR2 of AP17R1-2H2-Z1-R1-27A2 27 CDR3 ofAP17R1-2H2-Z1-R1-27A2 28 CDR1 of AP17R1-2H2-Z1-R1-29B2 29 CDR2 ofAP17R1-2H2-Z1-R1-29B2 30 CDR3 of AP17R1-2H2-Z1-R1-29B2 31 CDR1 ofAP17R1-2H2-Z1-R1-29B6 32 CDR2 of AP17R1-2H2-Z1-R1-29B6 33 CDR3 ofAP17R1-2H2-Z1-R1-29B6 34 CDR1 of AP17R1-2H2-Z1-R1-30D3 35 CDR2 ofAP17R1-2H2-Z1-R1-30D3 36 CDR3 of AP17R1-2H2-Z1-R1-30D3 37 Full-lengthsequence of AP17R1-2H2 38 Full-length sequence of AP17R1-2H2-Z1 39Full-length sequence of AP17R1-2H2-Z1-R1-4B2 40 Full-length sequence ofAP17R1-2H2-Z1-R1-4D8 41 Full-length sequence of AP17R1-2H2-Z1-R1-6E1 42Full-length sequence of AP17R1-2H2-Z1-R1-14A1 43 Full-length sequence ofAP17R1-2H2-Z1-R1-14F1 44 Full-length sequence of AP17R1-2H2-Z1-R1-14B345 Full-length sequence of AP17R1-2H2-Z1-R1-14F3 46 Full-length sequenceof AP17R1-2H2-Z1-R1-27A2 47 Full-length sequence ofAP17R1-2H2-Z1-R1-29B2 48 Full-length sequence of AP17R1-2H2-Z1-R1-29B649 Full-length sequence of AP17R1-2H2-Z1-R1-30D3 50 Nucleotide sequenceencoding AP17R1-2H2 51 Nucleotide sequence encoding AP17R1-2H2-Z1 52Nucleotide sequence encoding AP17R1-2H2-Z1-R1-4B2 53 Nucleotide sequenceencoding AP17R1-2H2-Z1-R1-4D8 54 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-6E1 55 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-14A1 56 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-14F1 57 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-14B3 58 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-14F3 59 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-27A2 60 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-29B2 61 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-29B6 62 Nucleotide sequence encodingAP17R1-2H2-Z1-R1-30D3

EXAMPLES

The present invention, thus generally described, will be understood morereadily by reference to the following Examples, which are provided byway of illustration and are not intended to be limiting of the instantinvention. The Examples are not intended to represent that theexperiments below are all or the only experiments performed.

Example 1 Preparation of Materials 1. Preparation of Materials 1.1Commercial Materials

Information on the commercially available materials used in the examplesare provided in Table 1.

TABLE 1 Commercial materials Materials Vendor Cat. FITC mouse anti-humanCD279 Ab eBioscience Cat. #11-9969-42 FITC goat anti-human IgG FcBethlyl Cat. #A80-204F PE goat anti-mouse IgG Fc Abcam Cat. #ab98742R-PE goat anti-human IgG Fc Jackson Immuno Cat. #109-115-098 Research PEgoat anti-mouse IgG Fc Abcam Cat. #Ab98742 SA-PE eBioscience Cat.#12-4317 HRP goat anti-human IgG Fc Bethyl Cat. #A80-304PStreptavidin-HRP Invitrogen Cat. #SNN1004 Ficoll-Paque ™ PLUS GEHealthcare Cat. #17-1440-02 Human Monocyte Enrichment kit STEMCELL Cat.#19059 Human CD4⁺ T Cell Enrichment kit STEMCELL Cat. #19052 Recombinanthuman GM-CSF Amoytop Biotech Cat. #S10980039 Recombinant human IL-4 R&DCat. #204-IL-010 Standard recombinant human IFN-γ PeproTech Cat. #300-02Human IFN-γ capture antibody Pierce Cat. #M700A Human IFN-γ detectionantibody Pierce Cat. #M701B SA-HRP Invitrogen Cat. #SNN1004 Human PD-1,His tag Sino Biological Cat. #10377-H08H Cynomolgus PD-1, His tagAcrobioSystem Cat. #PDl-C5223 Cynomolgus PD-1, hFc tag Sino BiologicalCat. #90311-C02H Mouse PD-1, His tag Sino Biological Cat. #50124-M08HHuman PD-L2, His tag Sino Biological Cat. #10292-H08H Human BTLA, Histag AcrobioSystems Cat. #BTA-H52E0 Cynomolgus PD-1, His tag R&D SystemsCat. #85509-PD

1.2 Material Code

The codes or abbreviation for the materials including benchmarkantibodies, the extracellular domains and the cells are summarized inTable 2.

TABLE 2 Material Code Material Name Code or Abbreviation Benchmarkantibody 1 (Opdivo ®, Nivolumab) BMK1, BMK1.IgG4, W305-BMK1.IgG4 orW305-BMK1.hIgG4 Benchmark antibody 3 BMK3 or BMK3.IgG4, W305-BMK3.hIgG4,or W305-BMK3.hIgG4K Human IgG4 isotype control hIgG4 Isotype, Isotype,human IgG4 Human PD-1 extracellular domain, mFc tag hPro1.ECD.mFc MousePD-1 extracellular domain, mFc tag mPro1.ECD.mFc Human PD-L1extracellular domain, mFc tag hProL1.ECD.mFc Mouse PD-L1 extracellulardomain, mFc tag mProL1.ECD.mFc Human CD28 extracellular domain, mFc taghCD28.ECD.mFc Human CTLA-4 extracellular domain, His tag hCTLA-4.ECD.HisHuman ICOS extracellular domain, mFc tag hICOS.ECD.mFc Human PD-1extracellular domain, His tag hPro1.ECD.His Mouse PD-1 extracellulardomain, His tag mPro1.ECD.His Human PD-L2 extracellular domain, His taghPro1L2.ECD.His Cynomolgus PD-1 extracellular domain, hFc tagcynoPro1.ECD.hFc Cynomolgus PD-L1 extracellular domain, hFc tagcynoProL1.ECD.hFc Cynomolgus PD-1 extracellular domain, His tagcynoPro1.ECD.His Human BTLA extracellular domain, His hBTLA.ECD.HisHuman PD-1-expressing CHO-S cell CHO-S.hPro1.C6 Mouse PD-1-expressing293F cell 293F.mPro1.B4 W3056 lead antibody (VHH-Fc(hIgG4) fusion)W3056-AP17R1-2H2-Z1-R1- 14A1-FC(IgG4.SP) W3056 lead parental antibody(VHH) AP17R1-2H2 W3056 lead parental antibody (VHH-Fc(hIgG4) fusion)AP17R1-2H2-Fc(IgG4), AP17R1-2H2-FC(IgG4.SP),W3056-AP17R1-2H2-FC(IgG4.SP)

2. Production of Antigens

DNA sequences encoding the antigens hPro1.ECD (Genbank Accession No.NP_005009.2), hProL1.ECD (Genbank Accession No. NP_054862.1),hCTLA-4.ECD (Genbank Accession No. NP_005205.2), hCD28.ECD (GenbankAccession No. NP_006130.1), hICOS.ECD (Genbank Accession No.NP_036224.1) and mPro1ECD (Genbank Accession No. NP_032824.1),mProL1.ECD (Genbank Accession No. NP_068693.1) were synthesized inSangon Biothech (Shanghai, China), and then subcloned into modifiedpcDNA3.3 expression vectors with different tag (such as 6×his, human Fc,or mouse Fc) at C-terminus. The resultant expression vectors werefurther purified. Expi293 cells (Invitrogen-A14527) were transfectedwith the purified expression vectors. The transfected cells werecultured for 5 days and supernatant was collected for proteinpurification using Ni-NTA column (GE Healthcare, 175248) or Protein Acolumn (GE Healthcare, 175438) or Protein G column (GE Healthcare,170618). The obtained antigens were quality controlled by SDS-PAGE andSEC size exclusion chromatography, and then stored at −80° C.

3. Production of Benchmark Antibodies

DNA sequences encoding the variable region of ant-PD-1 antibodies weresynthesized in Sangon Biothech (Shanghai, China), and then subclonedinto modified pcDNA3.3 expression vectors with constant region of humanIgG4 (S228P). The heavy and light chain variable region sequences ofBMK1 are the same as the approved anti-PD-1 antibody Opdivo (thesequence of clone 5C4 in PCT application WO2006/121168A1). The heavy andlight chain variable region sequences of BMK3 antibody are the same asthe sequence of clone 1B8 in US Patent No. US8168757B2.

Plasmids containing the DNA sequences encoding the heavy chain and lightchain regions of the respective anti-PD-1 antibodies were co-transfectedinto Expi293 cells. The transfected cells were cultured for 5 days andsupernatant was collected for protein purification using Protein Acolumn (GE Healthcare, 175438) or Protein G column (GE Healthcare,170618). The obtained antibodies were analyzed by SDS-PAGE and SEC, andthen stored at −80° C.

3. Establishment of Stable Cell Lines

Using Lipofectamine 2000, CHO-S or 293F cells were transfected with theexpression vector containing gene encoding full length human PD-1 ormouse PD-1. Cells were cultured in medium containing proper selectionpressure. Human PD-1 high expression stable cell line(WBP305.CHO-S.hPro1.C6) and mouse PD-1 high expression stable cell line(WBP305.293F.mPro1.B4) were obtained by limiting dilution.

EXAMPLE 2 Production of VHH and Chimeric VHH-Fc (hIgG4) Protein 1.Immunization

To induce a humoral immune response directed towards PD-1 in camelidanimals, the animals were subcutaneously injected with human and/ormouse PD-1 ECD proteins for 7 to 9 doses at 1 to 3 week intervals. Thedose ranged from 50 ug to 200 ug per injection.

2. Serum Titer Detection

After immunization, the anti-PD-1 specific antibody serum titer wasdetermined by ELISA. For ELISA test, ELISA plates (Nunc, Rochester,Minn., USA) were coated with 1 μg/ml of recombinant his tagged humanPD-1 ECD protein and mouse PD-1 ECD protein, respectively, and incubatedovernight at 4° C. After blocking and washing, serial dilutions ofpre-immune and immune sera were added and incubated at room temperaturefor 2 h, then followed by goat anti-L1 ama IgG-HRP (Novas Biologicals,Littleton, Colo., USA) at room temperature for 1 h. After washing, TMBsubstrate (Invitrogen, Carlsbad, Calif., USA) was added and the reactionwas stopped by 2M HCl. The absorbance at 450 nm was read using amicroplate reader (Molecular Device, Sunnyvale, Calif., USA).

3. Phage Library Construction

50 ml blood samples were collected at 6-7 days after the last twoinjections, respectively. Peripheral blood mononuclear cells (PBMCs)were purified by density gradient centrifugation on Ficoll-Paque PLUS(GE Healthcare, Little Chalfont, UK), resulting in the isolation ofapproximately 8×10⁷ PBMCs. Total RNA was extracted from the PBMCs andtranscribed into cDNA using an oligo-dT primer and SuperScript IIIFirst-Strand Synthesis SuperMix System (Invitrogen, Carlsbad, Calif.,USA) according to the manufacturers' recommendations.

The cDNA was purified and then used as template to amplify therepertoire of Ig heavy chain-encoding gene segments with the use ofsignal peptide domain specific primers and CH2 domain specific primers.This amplification resulted in PCR fragments of approximately 900 bp(representing conventional IgGs) and 700 bp (representing heavy-chainIgGs that lack a CH1 domain). The two classes of heavy chain encodinggenes were then size-separated on agarose gels and the genes encodingheavy-chain only IgG were purified by QlAquick Gel Extraction Kit(Qiagen, Hilden, Germany). The purified fragments were used as templateto amplify the VHH repertoire with the use of framework1 (FR1) andframework4 (FR4) specific primer pairs. This amplification procedureintroduced Sfi I restriction site at the 5′ end of FR1 and a Not Irestriction site at the 3′ end of FR4. The repertoire of PCR-amplifiedVHH genes of about 300-400 bp was loaded on agarose gels and purified byQlAquick Gel Extraction Kit. The purified fragments were then cut withSfi I and Not I and purified by QlAquick PCR Purification Kit (Qiagen,Hilden, Germany). The VHH gene fragments were finally ligated inphagemid vector pFL249 and electrotransformed into E. coli TG1. Aftertransformation, the TG1 cells were cultured in SOC medium with shakingat 200 rpm for 1 h, then the E. coli TG1 were plated onto platescontaining solid 2YT medium supplemented with 100 μg/mL Cab and 1% (w/v)glucose, and cultured at 37° C. overnight. The next day, the colonieswere scraped into liquid 2YT medium supplemented with 1/3 (v/v) of 80%glycerol and were stored at −80° C.

4. Phage Display Selection of Anti-PD-1 Specific VIM Fragments

To select VHH fragments that would effectively bind to PD-1, the methodsof protein panning and cell panning were employed.

For the protein panning, 20 μg of recombinant his tagged human and mousePD-1 ECD protein were immobilized in 5 ml immune tube (Nunc, Rochester,Minn., USA) overnight at 4° C. with shaking at 400 rpm, respectively. Onthe next day, after washing away unbound protein, the tube was blockedwith 10% skim milk for 1 h at 25° C. Approximately 10¹² cfu phages fromthe immune phage libraries added into non-coated immune tube blockedwith 10% skim milk to deplete the non-specifically bound phage, then thetreated phages were added into the tube and incubated at 25° C. for 2 h.After extensive washing with PBST, the nonspecifically adsorbed phageswere discarded and the target specifically bound phages were eluted byGlycine-HCl (pH2.2) and then neutralized by 1M Tris-HCl (pH8.0) forinfection of exponentially growing TG1 cells.

The infected TG1 cells were plated on 2YT agar plates containing 2%(w/v) glucose and 100 μg/ml ampicillin and cultured overnight at 37° C.On the next day, the colonies were scraped off the plate with 3 ml 2YTand frozen at −80° C. by adding in 1/3 (v/v) 80% glycerol. The scrapedbacteria libraries were inoculated into 2YT-Carb containing 100 μg/mlampicillin, and were infected with helper phage M13Ko7 in 2YT mediumwith 50 μg/ml kanamycin and 1 mM IPTG for phage rescue and used as inputfor the next round of panning.

For the cell panning, pre-incubation of 10¹² cfu phages with 2×10⁶-1×10⁷CHO-S or 293F cells was performed for depletion of non-specific boundphage particles. Then the treated phages were incubated with 2×10⁶-1×10⁷PD-1 transfected CHO-S or 293F cells at 4° C. for 1 h with tumbling at12 rpm. The cells were washed with ice cold 5% FBS-PBS and the cellbound phage particles were eluted as described above. For panning byDynabeads, about 10¹² cfu phages were first incubated with 200 μlDynabeads M-280 Streptavidin (Invitrogen, Carlsbad, Calif., USA) fordepletion of non-specific bound phage particles, then the treated phageswere incubated with another 200 μDynabeads saturated with 20 μgbiotinylated PD-1 ECD proteins at RT for 1 h with gentle mixing. Afterintensive washing, the phage that bound to beads were eluted asdescribed above.

5. VIM Protein Expression and Screening

After desired panning steps, phage infected TG1 cell colonies grown onthe plates were scraped and pFL249 phagemid containing VHH fragmentswere extracted. The VHH fragments were cloned by digestion of pFL249plasmids with Sfi I and Not I and then ligated into expression vectorpETbac containing genes of hexa-histidine- and c-Myc-tag. The ligationproducts were transformed into E. coli BL21 (DE3) competent cells andthen cultured in ZYM-5052 medium at 25° C. for 48 h with shaking at230rpm. Then the bacterial culture supernatants were collected for ELISAor FACS tests.

ELISA was used as the first screening method to test the binding of VHHto human PD-1 ECD protein. Briefly, 96-well plates (Nunc, Rochester, MN,USA) were coated with recombinant his tagged human and mouse PD-1 ECDprotein overnight at 4° C. After blocking and washing, the BL21 E. colisupernatants were transferred to the coated plates and incubated at roomtemperature for 1 h. The plates were then washed and subsequentlyincubated with secondary antibody Goat Anti-c-Myc-HRP (Bethyl,Montgomery, Tex., USA) for 1 h. After washing, TMB substrate was addedand the reaction was stopped by 2M HCl. The absorbance at 450 nm wasread using a microplate reader (Molecular Device, Sunnyvale, Calif.,USA).

In order to confirm the native binding of anti-PD-1 VHH onconformational PD-1 molecules expressed on cell membrane, flow cytometryanalysis was performed with human PD-1 transfected CHO-S cells and mousePD-1 transfected 293F cells. The parental CHO-S or 293F cell line wereused as negative control. The cells were firstly incubated with the E.coli culture supernatant samples in 96-well U-bottom plates (BD,Franklin Lakes, N.J., USA) at a density of 1×10⁵ cells/well at 4° C. for1 h, then with a secondary antibody Goat Anti-c-Myc-PE (Bethyl,Montgomery, Tex., USA) at 4° C. for 30 min. 2 times of washings wereapplied between each steps and the cells were resuspended in 1× PBS/1%BSA for flow cytometery analysis (IntelliCyt, Albuquerque, N. Mex.,USA).

6. Sequencing

The positive E. coli clones selected by ELISA and FACS screening weresent to Biosune (Shanghai, China) for nucleotide sequencing of VHH gene.The sequencing results were analyzed using CLC Main Workbench (Qiagen,Hilden, Germany).

One lead antibody was VHH named “AP17R1-2H2”. The sequence informationthereof is provided in Table A and the sequence listing. This antibodyalso served as the “parent” for optimization (including humanization andaffinity maturation). The humanization and affinity maturation will bedescribed in more details in Example 3 below.

7. VIM Production

The BL21 E. coli clones harboring VHH gene were cultured in 40 ml ofZYM-5052 medium at 25° C. for 48 h with shaking at 230 rpm. Theexpression of his- and c-Myc-tag fused VHH protein in BL21 supernatantwas confirmed by SDS-PAGE, and then purified using Ni-NTA column. Thepurity of VHH was determined by SEC-HPLC. For low supernatant expressionclones, ultrasonic (Scientz, Ningbo, China) breaking E. coli cells wasused to release soluble VHH proteins.

8. Chimeric (hIgG4) Protein Production

The clones of interest were converted to VHH-Fc (hIgG4) fusionantibodies. Briefly, the VHH genes were PCR amplified from the pET-bacvectors using VHH-specific cloning primers containing appropriaterestriction sites then cloned by fusion into a modified expressionpcDNA3.3 vector containing Fc of human hIgG4. S228P to createcorresponding clones of VHH-Fc (hIgG4. SP) chimeric antibody. 293F orExpi293 cells were transiently transfected with the vector for antibodyexpression. The cell culture supernatants containing antibodies wereharvested and purified using Protein A chromatography.

EXAMPLE 3 Antibody Optimization 1. Humanization

VHHs with high affinity and specificity to PD-1 were selected forhumanization. “Best Fit” approach was used to humanize VHH chains.

Amino acid sequences of VHH framework regions were blasted against humangermline V-gene database, and humanized VHH sequences were generated byreplacing human CDR sequences in the top hit with VHH CDR sequencesusing Kabat CDR definition. Certain residues in the framework regionwere back-mutated to VHH in order to maintain the affinity. Humanizedgenes were back-translated, codon optimized for mammalian expression,and synthesized by GENEWIZ. These genes were re-amplified with cloningprimers containing appropriate restriction sites and cloned into amodified pcDNA3.3 vector to express humanized VHH-Fc (hIgG4.SP). Aftertesting on PD-1 binding using SPR, the variants with proper affinitywere selected as humanized antibody leads. The VHH-Fc (hIgG4) form forthe parental antibody “AP17R1-2H2” is named as “AP17R1-2H2-Fc(IgG4)” or“W3056-AP17R1-2H2-FC(IgG4. SP).”

2. Affinity Maturation

Each amino acid of three complementary-determining regions (CDR1, CDR2,and CDR3) of parental clone was individually mutated to other 20 aminoacids using a site-directed mutagenesis method. DNA primers containing aNNS codon encoding twenty amino acids were used to introduce mutation toeach targeted CDR position. The individual degenerate primers ofphosphorylated were used in site-directed mutagenesis reactions. 200 ngof the reaction products was electroporated into BL21 and expressed.

The mutant clones were screened by using competitive ELISA assay.Briefly, 96-well Maxisorp Immunopla was coated with 0.5 ug/ml anti-c-Mycantibody in coating buffer (200 mM Na2CO3/NaHCO3) at pH 9.2 overnight at4° C. The next day, the plate was blocked with Casein for 1 h at roomtemperature. After blocking, E. coli supernatants of above mutant clonesand parental VHH (i.e., AP17R1-2H2) clone were added to the plate andincubated at room temperature for 1 h. After washing the plate, thepre-mixture of hPro1.hFc-biotin (0.25 ug/ml) and AP17R1-2H2-FC (IgG4.sp)(0.25 ug/ml) were added to the well and incubated for 1 h at roomtemperature. This was followed by incubation with Streptavidin-HRPconjugate for 1 h at room temperature. HRP activity was detected withTMB substrate and the reaction was stopped with 2 M HCl. Plates wereread at 450 nm. The clones exhibiting an optical density (OD) signal at450 nm greater than 1.5-fold of that of the parental VHH (i.e.,AP17R1-2H2) clone were picked and sequenced. These mutant clones werere-expressed in BL21 and purified. Surface plasmon resonance (SPR) wasused for detecting the affinity of the mutants. Upon humanization andaffinity maturation, a series of variants were obtained.

3. K_(D) Ranking by SPR

To screen WBP3056 VHH antibodies from affinity maturation, koff tohPro1.ECD.hFc, mPro1.ECD.hFc and cynoPro1.ECD.hFc (AcrobioSystems) wasdetected by SPR assay using Biacore 8K. hPro1.ECD.hFc, mPro1.ECD.hFc orcynoPro1.ECD.hFc (AcrobioSystems) was captured on an anti-human IgG Fcantibody immobilized CMS sensor chip. Each VHH antibody was injectedover the sensor chip at a flow rate of 30 uL/min for an associationphase of 120 s, followed by 150 s dissociation. The association anddissociation curve was fit to a 1:1 model using Langmuir analysis. Theresults are shown in Table 3.

TABLE 3 hPro1.ECD.hFc mPro1.ECD.hFc cynoPro1.ECD.hFc Analyte ka kd KD kakd KD ka kd KD (VHH protein) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) (1/Ms)(1/s) (M) AP17R1-2H2-Z1-R1-4B2 4.50 × 10⁵ 4.13 × 10⁻³ 9.19 × 10⁻⁹ 2.92 ×10⁵ 1.01 × 10⁻² 3.46 × 10⁻⁸ 3.45 × 10⁵ 8.79 × 10⁻³ 2.55 × 10⁻⁸AP17R1-2H2-Z1-R1-4D8 4.17 × 10⁵ 3.96 × 10⁻³ 9.49 × 10⁻⁹ 2.84 × 10⁵ 9.55× 10⁻³ 3.37 × 10⁻⁸ 3.20 × 10⁵ 8.33 × 10⁻³ 2.60 × 10⁻⁸AP17R1-2H2-Z1-R1-6E1 4.39 × 10⁵ 3.28 × 10⁻³ 7.48 × 10⁻⁹ 2.87 × 10⁵ 1.46× 10⁻² 5.08 × 10⁻⁸ 3.41 × 10⁵ 7.54 × 10⁻³ 2.21 × 10⁻⁸AP17R1-2H2-Z1-R1-14A1 5.43 × 10⁵ 1.12 × 10⁻³ 2.06 × 10⁻⁹ 3.81 × 10⁵ 4.70× 10⁻³ 1.23 × 10⁻⁸ 4.24 × 10⁵ 2.13 × 10⁻³ 5.04 × 10⁻⁹AP17R1-2H2-Z1-R1-14F1 4.11 × 10⁵ 1.35 × 10⁻³ 3.28 × 10⁻⁹ 2.80 × 10⁵ 2.18× 10⁻³ 7.80 × 10⁻⁹ 3.09 × 10⁵ 2.77 × 10⁻³ 8.98 × 10⁻⁹AP17R1-2H2-Z1-R1-14B3 4.89 × 10⁵ 2.49 × 10⁻³ 5.09 × 10⁻⁹ 3.44 × 10⁵ 5.96× 10⁻³ 1.73 × 10⁻⁸ 3.66 × 10⁵ 4.84 × 10⁻³ 1.32 × 10⁻⁸AP17R1-2H2-Z1-R1-14F3 4.65 × 10⁵ 1.09 × 10⁻³ 2.35 × 10⁻⁹ 2.70 × 10⁵ 7.32× 10⁻³ 2.71 × 10⁻⁸ 3.44 × 10⁵ 2.47 × 10⁻³ 7.18 × 10⁻⁹AP17R1-2H2-Z1-R1-27A2 3.87 × 10⁵ 3.31 × 10⁻³ 8.56 × 10⁻⁹ 2.65 × 10⁵ 8.94× 10⁻³ 3.37 × 10⁻⁸ 3.02 × 10⁵ 7.45 × 10⁻³ 2.46 × 10⁻⁸AP17R1-2H2-Z1-R1-29B2 4.06 × 10⁵ 3.47 × 10⁻³ 8.54 × 10⁻⁹ 2.64 × 10⁵ 1.48× 10⁻² 5.58 × 10⁻⁸ 3.18 × 10⁵ 8.17 × 10⁻³ 2.57 × 10⁻⁸AP17R1-2H2-Z1-R1-29B6 4.25 × 10⁵ 2.98 × 10⁻³ 7.01 × 10⁻⁹ 2.77 × 10⁵ 1.08× 10⁻² 3.91 × 10⁻⁸ 3.23 × 10⁵ 6.46 × 10⁻³ 2.00 × 10⁻⁸AP17R1-2H2-Z1-R1-30D3 4.16 × 10⁵ 2.82 × 10⁻³ 6.79 × 10⁻⁹ 2.77 × 10⁵ 1.18× 10⁻² 4.25 × 10⁻⁸ 3.25 × 10⁵ 6.24 × 10⁻³ 1.92 × 10⁻⁸ AP17R1-2H2-Z1 3.07× 10⁵ 3.92 × 10⁻³ 1.27 × 10⁻⁸ 2.05 × 10⁵ 1.36 × 10⁻² 6.62 × 10⁻⁸ 2.37 ×10⁵ 8.63 × 10⁻³ 3.64 × 10⁻⁸

In Table 3, it is demonstrated that, after affinity maturation, theaffinity maturated VHHs bind to hPro1.ECD.hFc, mPro1.ECD.hFc, andcynoPro1.ECD.hFc, respectively, with better affinities than that beforeaffinity maturation (e.g. AP17R1-2H2-z1, the humanized AP17R1-2H2).

Sequence information on the VHH proteins listed in Table 3 are providedin above Table A and the accompanying sequence listing.

EXAMPLE 4 In Vitro Characterization Binding to human, mouse, andCynomolgus monkey PD-1 as Measured by FACS 1.1 Human PD-1 Binding

The cells CHO-S.hPro1.C6 (2×10⁵ cells/well) were incubated with variousconcentrations of anti-PD-1 antibodies (4-fold serially diluted from133.3 nM to 0.008 nM) at 4° C. for 1 hour. After washing with1×PBS/1%BSA, the secondary antibody PE-labeled goat anti-human IgG wasapplied and incubated with cells at 4° C. for 1 hour. Anti-human PD-1antibodies BMK1 and BMK3 were used as positive control. Human IgG4isotype antibody was used as negative control. The cells were thenwashed and resuspended in 1×PBS/1%BSA. MFI of the cells was measured bya flow cytometer (BD) and analyzed by FlowJo (version 7.6.1). Data wereshown in FIG. 1 and Table 4.

TABLE 4 Binding EC₅₀ of to human PD-1 by FACS Antibody EC₅₀ (nM)W3056-AP17R1-2H2-FC(IgG4.SP) 1.872W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) 0.612 WBP305-BMK1.hIgG40.409-0.749 WBP305-BMK3.hIgG4 3.103

As shown in FIG. 1 and Table 4, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) effectively bound to cell surface human PD-1 in adose-dependent manner.

1.2 Mouse PD-1 Binding

The cells 293F.mPro1.B4 (2×10⁵ cells/well) were incubated with variousconcentrations of anti-PD-1 antibodies (3-fold serially diluted from133.3 nM to 0.008 nM) at 4° C. for 1 hour. After washing with 1×PBS/1%BSA, the secondary antibody PE-labeled goat anti-human IgG was appliedand incubated with cells at 4° C. for 1 hour. Human IgG4 isotypeantibody was used as negative control. The cells were then washed andre-suspended in 1×PBS/1% BSA. MFI of the cells was measured by a flowcytometer and analyzed by FlowJo (version 7.6.1). Data were shown inFIG. 2 and Table 5.

TABLE 5 Binding EC50 to mouse PD-1 by FACS Antibody EC₅₀ (nM)W3056-AP17R1-2H2-FC (IgG4.SP) 4.77W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) 4.257 WBP305-BMK1.hIgG4 NAWBP305-BMK3.hlgG4 NA

As shown in FIG. 2 and Table 5, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) and the antibodyAP17R1-2H2-FC(IgG4) effectively bound to cell surface mouse PD-1 in adose-dependent manner.

1.3 Cynomolgus Monkey PD-1 Binding

Cynomolgus monkey PD-1 (GenBank Accession No. NP_001271065.1) transienttransfected 293F cells (2×10⁵ cells/well) were incubated with variousconcentrations of anti-PD-1 antibodies (3-fold serially diluted from133.4 nM to 0.008 nM) at 4° C. for 1 hour. After washing with 1×PBS/1%BSA, the secondary antibody PE-labeled goat anti-human IgG was appliedand incubated with cells at 4° C. for 1 hour. Anti-human PD-1 antibodiesBMK1 and BMK3 were used as positive control. Human IgG4 isotype antibodywas used as negative control. The cells were then washed andre-suspended in 1×PBS/1%BSA. MFI of the cells was measured by a flowcytometer and analyzed by FlowJo (version 7.6.1). Data were shown inFIG. 3 and Table 6.

TABLE 6 Binding EC50 to cyno PD-1 by FACS in two separated experimentsEC₅₀ (nM) EC₅₀ (nM) Antibody (experiment 1) (experiment 2)W3056-AP17R1-2H2-FC (IgGASP) 0.98 / W3056-AP17R1-2H2-Z1-R1-14A1- / 1.975FC(IgG4.SP) W305-BMK1.hIgG4 0.55 1.981 W305-BMK3.hIgG4 1.03 2.293

As shown in FIG. 3 and Table 6, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) effectively bound to cell surface cynomolgus PD-1 ina dose-dependent manner.

2. Blocking of PD-L1 or PD-L2 Binding to PD1 2.1 Human PD-1/PD-L1Blocking as Measured by FACS

The human PD-1 transfected CHO-S.hPro1.C6 cells were transferred into96-well U-bottom plates at a density of 2×10⁵ cells/well. Variousconcentrations of antibodies (4-fold serially diluted from 133.3 nM to0.008 nM) and constant concentration of mouse Fc tagged PD-L1 ECDprotein (hProL1.ECD.mFc) (5 μg/mL) were pre-mixed and incubated withcells at 4° C. for 1 hour. After washing with 1×PBS/1% BSA, thesecondary antibody PE-labeled goat anti-mouse IgG was applied andincubated with cells at 4° C. for 1 hour. The cells were then washed andresuspended in 1×PBS/1% BSA. MFI of the cells was measured by a flowcytometer and analyzed by FlowJo (version 7.6.1). Data were shown inFIG. 4 and Table 7.

TABLE 7 Blocking human PD-L1 binding to cell surface human PD-1 AntibodyIC50 (nM) W3056-AP17R1-2H2-Fc (IgG4.SP) 1.000W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) 0.655 WBP305-BMK1.hIgG4 0.544WBP305-BMK3.hIgG4K 1.763

As shown in FIG. 4 and Table 7, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) are effective at blocking the binding of human PD-L1to cell surface human PD-1 in a dose-dependent manner.

2.2. Mouse PD-1/PD-L1 Blocking as Measured by FACS

The mouse PD-1 transfected 293F.mPro1.B4 cells were transferred into96-well U-bottom plates at a density of 2×10⁵ cells/well. Variousconcentrations of anti-PD-1 antibodies (3-fold serially diluted from1334 nM to 0.07 nM) and constant concentration of mouse Fc tagged mousePD-L1 ECD protein (mProL1.ECD.mFc) (5 μg/mL) were pre-mixed andincubated with cells at 4° C. for 1 hour. After washing with 1×PBS/1%BSA, the secondary antibody PE-labeled goat anti-mouse IgG was appliedand incubated with cells at 4° C. for 1 hour. The cells were then washedand resuspended in 1×PBS/1% BSA. MFI of the cells was measured by a flowcytometer and analyzed by FlowJo (version 7.6.1). Data were shown inFIG. 5 and Table 8.

TABLE 8 Blocking mouse PD-L1 binding to cell surface mouse PD-1 AntibodyIC50 (nM) W3056-AP17R1-2H2-FC(IgG4.SP) 22.09W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) 10.19 WBP305-BMK1.hIgG4 NAWBP305-BMK3.hIgG4K NA

As shown in FIG. 5 and Table 8, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) are effective at blocking the binding of mouse PD-L1to cell surface PD-1 in a dose-dependent manner.

2.3 Human PD-1/PD-L2 Blocking as Measured by ELISA

Plates were pre-coated with 1 μg/mL, 100 μL per well of human PD-1 ECD(hPro1.ECD.mFc) at 4° C. overnight. After 1-hour blocking using 200 μLof 1×PBS/2% BSA, constant concentration of his tagged human PD-L2 ECD(hPro1L2.ECD.His) and various concentrations of testing antibodies(3-fold serially diluted from 66.7 nM to 0.003 nM) were pre-mixed andadded to the plates. After 2-hour incubation at ambient temperature, thebinding of the ligand to the immobilized protein was detected byHRP-labeled goat anti-His antibody. The color was developed bydispensing 100 μL of TMB substrate, and then stopped by 100 μL of 2NHCl. The absorbance was read at 450 nm and 540 nm using a microplatespectrophotometer. Data were shown in FIG. 6 and Table 9.

TABLE 9 Blocking human PD-L2 binding to immobilized human PD-1 in twoseparated experiments IC50 (nM) IC50 (nM) Antibody (experiment 1)(experiment 2) W3056-AP17R1-2H2-FC(IgG4.SP) 2.02 /W3056-AP17R1-2H2-Z1-R1-14A1- / 0.8856 FC(IgG4.SP) WBP305-BMK1.hIgG4 0.950.5338 WBP305-BMK3.hIgG4K 2.19 1.499

As shown in FIG. 6 and Table 9, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) are effective at blocking the binding of human PD-L2to immobilized PD-1 in a dose-dependent manner.

2.4 Cynomolgus Monkey PD-1/PD-L1 Blocking as Measured by ELISA

Plates were pre-coated with 1 μg/mL, 100 μL per well of cyno PD-1 ECDprotein (cynoPro1.ECD.hFc) at 4° C. overnight. After 1-hour blockingusing 200 μL of 1×PBS/2% BSA, constant concentration of biotinylatedcyno PD-L1 (cynoProL1.ECD.hFc.biotin) (2.5 μg/mL) and variousconcentrations of testing antibody, positive and negative control(3-fold serially diluted from 133.4 nM to 0.007 nM) were pre-mixed andadded to the plates. The plates were incubated at ambient temperaturefor 1 hour. The binding of ligand to the immobilized protein wasdetected by streptavidin-HRP. The color was developed by dispensing 100μL of TMB substrate, and then stopped by 100 μL of 2M HCl. Theabsorbance was read at 450 nm and 540 nm using a microplatespectrophotometer. Data were shown in FIG. 7 and Table 10.

TABLE 10 Blocking cyno PD-L1 binding to immobilized cyno PD-1 in twoseparated experiments IC50 (nM) IC50 (nM) Antibody (experiment 1)(experiment 2) W3056-AP17R1-2H2-FC(IgG4.SP) 1.30 /W3056-AP17R1-2H2-Z1-R1- / 0.8534 14A1-FC(IgG4.SP) WBP305-BMK1.hIgG4 0.470.5848 WBP305-BMK3.hIgG4K 14.21 14.59

As shown in FIG. 7 and Table 10, W3056 antibodies including the antibodyW3056-AP17R1 -2H2-Z1-R1-14A1 -FC(IgG4. SP) and the antibodyAP17R1-2H2-FC(IgG4) are effective at blocking the binding of cyno PD-L1to immobilized cyno PD-1 in a dose-dependent manner.

3. Cross-Family Protein Binding Assay as Measured by ELISA

Plates were pre-coated with 1 μg/mL, 100 μL per well of hPD-1.ECD.mFc,hCD28.ECD.mFc, hCTLA-4.ECD.His, hICOS.ECD.mFc or hBTLA.ECD.His at 4° C.overnight. After 1-hour blocking using 200 μL of 1×PBS/2% BSA, testingantibodies were added to the plates at a concentration of 100 nM. Theplates were incubated at ambient temperature for 1 hour. The binding ofthe antibodies to the immobilized proteins was detected by HRP-labeledgoat anti-human IgG antibody. The color was developed by dispensing 100μL of TMB substrate, and then stopped by 100 μL of 2N HCl. Theabsorbance was read at 450 nm and 540 nm using a microplatespectrophotometer. Data were shown in FIG. 8.

As shown in FIG. 8, W3056 antibodies including the antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) and the antibodyAP17R1-2H2-FC(IgG4) specifically bound to immobilized human PD-1 and didnot cross-react to human CD28, CTLA-4, ICOS and BTLA.

4. Epitope Binning by FACS

The human PD-1 transfected cells CHO-S.hPro1.C6 were transferred into96-well U-bottom plates at a density of 1-2×10⁵ cells/well. Serialdilutions of testing antibodies (4-fold serially diluted from 133.3 nMto 0.008 nM) were mixed with constant concentration of biotinylated BMK1(1 μg/mL) or biotinylated BMK3 (1 μg/mL), respectively. Then the mixturewas added to the cells in 96-well plates and incubated at 4° C. for 1hour. After washing with 1×PBS/1%BSA, the secondary antibody SA-PE wasapplied and incubated with cells at 4° C. for 1 hour. The cells werethen washed and resuspended in 1×PBS/1%BSA. MFI of the cells wasmeasured by a flow cytometer and analyzed by FlowJo. Data were shownFIG. 9.

As shown in FIG. 9, W3056 antibodies W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) and AP17R1-2H2 share similar epitope bin with BMK1 and BMK3.

5. Human Cell Based Functional Assays

One-way mixed lymphocyte reaction (one-way MLR) was used to test theagonistic effect of PD-1 antibodies on cytokine secretion of human CD4⁺T cells.

i) Cell isolation, cell culture and induction

Human PBMCs were freshly isolated from healthy donors using Ficoll-PaquePLUS gradient centrifugation. Isolated PBMCs were cultured in completeRPMI-1640 (containing 10% FBS and 1% PS) supplemented with 100 U/mLrecombinant human IL-2.

Human monocytes were isolated using Human Monocyte Enrichment Kitaccording to the manufacturer's instructions. Cell concentration wasadjusted to 2×10⁶ cells/mL in complete RPMI-1640 medium supplementedwith recombinant human GM-CSF at 800 U/mL and IL-4 at 50 ng/mL. Cellsuspension was seeded at 2.5 mL/well in 6-well plate. Cells werecultured for 5 to 7 days to differentiate into dendritic cells (DCs).Cytokines were replenished every 2-3 days by replacing half of the mediawith fresh media supplemented with cytokines.

Human CD4⁺ T cells were isolated using Human CD4⁺ T cell Enrichment kitaccording to the manufacturer's protocol.

ii) Mixed Lymphocyte Reaction

For human allogeneic MLR, purified CD4⁺ T cells were co-cultured withallogeneic immature DCs (iDCs). For human autologous MLR, PBMCs weretreated with CMV peptide for 5 days before CD4+ T cell isolation. On theday of assay, DCs were treated with CMV peptide for one hour, and thenco-cultured with autologous human CD4⁺ T cells.

MLR was set up in 96-well round bottom plates using complete RPMI-1640medium. CD4⁺ T cells, various concentrations of antibodies andallogeneic DCs were added to the plates at an appropriate ratio. Theplates were incubated at 37° C., 5% CO₂. After incubation for 3-5 days,the cytokine production or cell proliferation was detected.

iii) Cytokine Detection

Human IL-2 and IFN-γ release were measured by ELISA using matchedantibody pairs. Recombinant human IL-2 and IFN-γ were used as standards.The serial concentrations of IL-2 was 2 ng/ml, 1 ng/ml, 0.5 ng/ml, 0.25ng/ml, 0.125 ng/ml, 0.063 ng/ml, 0.031 ng/ml, 0.016 ng/ml, 0.008 ng/ml,and that of IFN-γ was 8 ng/ml, 4 ng/ml, 2 ng/ml, 1 ng/ml, 0.5 ng/ml,0.25 ng/ml, 0.125 ng/ml, 0.063 ng/ml, 0.031 ng/ml. The plates werepre-coated with capture antibody specific for human IL-2 or IFN-γ,respectively. After blocking, 100 μL of standards or samples werepipetted into each well and incubated for 2 hours at ambienttemperature. Following removal of the unbound substances, thebiotin-conjugated detecting antibody was added to the wells andincubated for one hour. Streptavidin-HRP was then added to the wells for30 minutes at ambient temperature. The color was developed by dispensing100 μL of TMB substrate, and then stopped by 100 μL of 2N HCl. Theabsorbance was read at 450 nm using a microplate spectrophotometer. Theconcentration of cytokine in supernatant was calculated from thestandard curve.

iv) Proliferation Detection

The number of viable cells in culture were determined by CellTiter-GloLuminescent Cell Viability Assay according to the manufacture'sinstruction.

As shown in FIGS. 10A and 10B W3056 antibodies includingW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) and AP17R14-2H2-Fc(IgG4)promoted IL-2 and IFN-γ production in a dose-dependent manner in humanallo-MLR reaction. FIGS. 11A and 11B show that W3056 antibodyAP17R1-2H2-Z1-R1-14A1-FC (IgG4.SP) promoted IFN-γ production and T cellproliferation in auto-MLR reaction.

v) Suppressive Treg Inhibition Assay

Human CD4⁺ T lymphocytes were enriched from PBMC of one healthy donor,and human monocytes were isolated from another healthy donor. RegulatoryT cells (Tregs) were isolated from human CD4⁺ T cells using HumanCD4⁺CD25^(HIGH) T Cell Isolation kit, and the remaining T lymphocyteswere CD4⁺CD25⁻ T cells. DCs were induced from monocyte as describedabove.

Ten thousand immature DCs, 1×10⁵ CD4⁺CD25⁻ T effector cells, 1×10⁵CD4⁺CD25⁺ Treg cells and 166.7 nM anti-PD-1 antibodies were incubated ina total volume of 200 μL in 96-well plates. The plates were kept at 37°C. in a 5% CO₂ incubator for 5 days. IFN-γ release and T cellsproliferation were examined. BMK1 and BMK3 were used as positivecontrol, and human IgG4 isotype antibody was used as negative control.Wells without Treg cells or anti-PD-1 antibody were also included ascontrols.

As shown in FIG. 12, the W3056 antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP), can reverse human Treg inducedT effector cell suppression, measured by IFN-γ production (FIG. 12A) andT cell proliferation (FIG. 12B)

vi). Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The human PD-1 transfected cell line WBP305-CHO-S.hPro1.C6 cells andvarious concentrations of testing antibodies were mixed in 96-wellplate, and PBMCs were added at the effector/target ratio of 50:1. Theplate was kept at 37° C. in a 5% CO₂ incubator for 4-6 hours. Targetcell lysis was determined by LDH-based cytotoxicity detection kit.Herceptin induced ADCC effect on SK-Br-3 cells was used as positivecontrol. The results indicated that W3056 antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC (IgG4.SP) did not induce ADCC effects onhuman PD-1 transfected cells, as shown in FIG. 13.

16. Affinity to Human, Mouse and Cyno PD-1 by Surface Plasmon Resonance(SPR)

Antibody binding affinity to human, mouse and cynomolgus PD-1 wasdetected using Biacore 8K. W3056-AP17R1-2H2-Z1-R1-14A1-FC (IgG4.SP) wascaptured on an anti-human Fc IgG antibody immobilized CMS sensor chip(GE). Human, mouse and cynomolgus PD-1 at different concentrations wereinjected over the sensor chip at a flow rate of 30 uL/min for anassociation phase of 120 s, followed by 150 s dissociation. The chip wasthen regenerated by 10 mM Glycine pH 1.5 after each binding cycle.

The sensorgrams of blank surface and buffer channel were subtracted fromthe test sensorgrams. The experimental data was fitted by 1:1 modelusing Langmiur analysis. Molecular weight of 40, 45 and 40 kDa was usedto calculate the molar concentration of analyte human, mouse andcynomolgus PD-1 respectively. The SPR results show that the affinitiesof W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) to human, cyno and mouse PD-1were 3.45E-09M, 8.53E-09M and 2.87E-08M, respectively (Table 11).

TABLE 11 Binding kinetic affinity tested by surface plasmon resonance(SPR) Binding to human PD-1 Binding to cyno PD-1 Binding to mouse PD-1ka kd KD ka kd KD ka kd KD Ligand (1/Ms) (1/s) (M) (1/Ms) (1/s) (M)(1/Ms) (1/s) (M) W3056-AP17R1-2H2- 2.48E+05 8.57E−04 3.45E−09 2.08E+051.77E−03 8.53E−09 1.47E+05 4.22E−03 2.87E−08 Z1-R1-14A1-FC(IgG4.SP)WBP305-BMK1.IgG4 4.05E+05 1.16E−03 2.87E−09 3.01E+05 6.72E−04 2.23E−09No/weak binding WBP305-BMK3.hIgG4K 1.77E+05 6.63E−04 3.74E−09 1.84E+055.17E−02 2.81E−07

7. Thermal Stability by DSF Assay

The DSF assay was performed using Real-Time Fluorescent Quantitative PCR(QuantStudio 7 Flex, Thermo Fisher Scientific). Briefly, 19 μL ofantibody solution was mixed with 1 μL of 62.5× SYPRO Orange solution(Invitrogen) and added to a 96-well plate (Biosystems). The plate washeated from 26° C. to 95° C. at a rate of 2° C./min, and the resultingfluorescence data were collected. The negative derivatives of thefluorescence changes with respect to different temperatures werecalculated, and the maximal value was defined as melting temperature Tm.If a protein has multiple unfolding transitions, the first two Tm werereported, named as Tm1 and Tm2. Tm1 is always interpreted as the formalmelting temperature Tm to facilitate comparisons between differentproteins. Data collection and Tm calculation were conductedautomatically by its operation software. Once the plot of negativederivatives of different temperatures was reported by the software(QuantStudio Real-Time PCR PCR Software v1.3). The DST result is shownin table 12 and DSF profile is shown in FIG. 14. W3056 antibodies havenormal DSF profiles and the Tm values are 60.3˜62° C.

TABLE 12 Thermal stability result by DSF W3056-AP17R1-2H2-W3056-AP17R1-2H2-Z1- Protein Name Fc(IgG4.SP) R1-14A1-FC(IgG4.SP)Isotype hIgG4 hIgG4 pI 6.4 6.4 Buffer PBS PBS Concentration 2.8 3.5(mg/ml) Tm(° C.) 62.0  60.3 

8.Human Serum Stability Assay

Human serum was freshly isolated from a healthy donor by centrifugation.The antibody was 10-fold diluted in human serum. Five aliquots of thesample were incubated at 37° C. Sample at day 0, day 1, day 4, day 7 andday 14 were collected and frozen in liquid nitrogen, respectively.

The human PD-1 transfected cell line WBP305-CHO-S.hPro1.C6 cells (1×10⁵cells/well) were incubated with various concentrations of serum treatedW3056 anti-PD-1 antibodies (3-fold serially diluted from 129.9 nM to0.0007 nM) at 4° C. for 1 hour. After washing with 1×PBS/1% BSA, thesecondary antibody PE-labeled goat anti-human IgG was applied andincubated with cells at 4° C. for 1 hour. Human IgG4 isotype antibodywas used as negative control. The cells were then washed and resuspendedin 1×PBS/1% BSA. MFI of the cells was measured by a flow cytometer (BD)and analyzed by FlowJo.

The results shown in FIG. 15 indicate that W3056 antibodyW3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP) was stable in human serum for atleast 14 days.

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments that have been described in detail herein. Rather, referenceshould be made to the appended claims as indicative of the scope andcontent of the invention.

1.-35. (canceled)
 36. A Programmed Death 1 (PD-1) binding moleculecomprising at least one immunoglobulin single variable domain (forexample, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, andwherein: a) CDR1 comprises an amino acid sequence which is at least 90%identical to SEQ ID NO: 13, CDR2 comprises an amino acid sequence whichis at least 90% identical to SEQ ID NO: 14, and CDR3 comprises an aminoacid sequence which is at least 80% identical to SEQ ID NO: 15; or b)CDR1 differs in amino acid sequence from SEQ ID NO: 13 by an amino acidaddition, deletion or substitution of not more than 2 amino acids; CDR2differs in amino acid sequence from SEQ ID NO: 14 by an amino acidaddition, deletion or substitution of not more than 2 amino acids;and/or CDR3 differs in amino acid sequence from SEQ ID NO: 3 by an aminoacid addition, deletion or substitution of not more than 2 amino acids.37. A Programmed Death 1 (PD-1) binding molecule comprising at least oneimmunoglobulin single variable domain (for example, VHH), wherein theVHH comprises CDR1, CDR2 and CDR3 selected from the group comprising:(a) CDR1 which is represented by DSIX₁SX₂VNMG, wherein X₁=D or Q, andX₂=M or L; (b) CDR2 which is represented by LIAX₃YITHYADFVKG, whereinX₃=N, T, Y, R or W; (c) CDR3 which is represented by RX₄IX₅X₆DY, whereinX₄=N or S, X₅=I, R or Y, and X₆=V or E.
 38. The PD-1 binding molecule ofclaim 36, wherein the PD-1 binding molecule is a PD-1 antagonist, forexample, an anti-PD-1 antibody.
 39. A Programmed Death 1 (PD-1) bindingmolecule comprising at least one immunoglobulin single variable domain(for example, VHH), wherein the VHH comprises CDR1, CDR2 and CDR3selected from the group comprising: (a) CDR1 with an amino acid sequenceas shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown inSEQ ID NO: 2, and CDR3 with an amino acid sequence as shown in SEQ IDNO: 3; (b) CDR1 with an amino acid sequence as shown in SEQ ID NO: 4,CDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and CDR3 withan amino acid sequence as shown in SEQ ID NO: 6; (c) CDR1 with an aminoacid sequence as shown in SEQ ID NO: 7, CDR2 with an amino acid sequenceas shown in SEQ ID NO: 8, and CDR3 with an amino acid sequence as shownin SEQ ID NO: 9; (d) CDR1 with an amino acid sequence as shown in SEQ IDNO: 10, CDR2 with an amino acid sequence as shown in SEQ ID NO: 11, andCDR3 with an amino acid sequence as shown in SEQ ID NO: 12; (f) CDR1with an amino acid sequence as shown in SEQ ID NO: 16, CDR2 with anamino acid sequence as shown in SEQ ID NO: 17, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 18; (g) CDR1 with an amino acidsequence as shown in SEQ ID NO: 19, CDR2 with an amino acid sequence asshown in SEQ ID NO: 20, and CDR3 with an amino acid sequence as shown inSEQ ID NO: 21; (h) CDR1 with an amino acid sequence as shown in SEQ IDNO: 22, CDR2 with an amino acid sequence as shown in SEQ ID NO: 23, andCDR3 with an amino acid sequence as shown in SEQ ID NO: 24; (i) CDR1with an amino acid sequence as shown in SEQ ID NO: 25, CDR2 with anamino acid sequence as shown in SEQ ID NO: 26, and CDR3 with an aminoacid sequence as shown in SEQ ID NO: 27; (j) CDR1 with an amino acidsequence as shown in SEQ ID NO: 28, CDR2 with an amino acid sequence asshown in SEQ ID NO: 29, and CDR3 with an amino acid sequence as shown inSEQ ID NO: 30; (k) CDR1 with an amino acid sequence as shown in SEQ IDNO: 31, CDR2 with an amino acid sequence as shown in SEQ ID NO: 32, andCDR3 with an amino acid sequence as shown in SEQ ID NO: 33; and (l) CDR1with an amino acid sequence as shown in SEQ ID NO: 34, CDR2 with anamino acid sequence as shown in SEQ ID NO: 35, and CDR3 with an aminoacid sequence as shown in SEQ ID NO:
 36. 40. The PD-1 binding moleculeof claim 36, wherein the VHH comprises (A) the amino acid sequence shownin any of SEQ ID NOs: 37-49; (B) an amino acid sequence which is atleast 85%, at least 90%, or at least 95% identical to any of SEQ ID NOs:37-49; or (C) an amino acid sequence with addition, deletion and/orsubstitution of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or10) amino acids compared with any of SEQ ID NOs: 37-49.
 41. The PD-1binding molecule of claim 36, wherein the PD-1 binding molecule is asingle-domain antibody, for example a heavy chain single-domainantibody; a chimeric antibody of VHH from a camelid animal and Fc domainof human IgG4; or a humanized antibody, or the VHH is fused to anothermolecule, for example, a Fc domain of an immunoglobin (for example,IgG), a fluorescent protein or a VHH with a distinct specificity. 42.The PD-1 binding molecule of claim 36, wherein the PD-1 binding moleculehas one or more of the following properties: (a) binds human PD-lwith aKD of 1×10⁻⁷ M or less; (b) inhibits binding of PD-L1 or PD-L2 to PD-1;(c) induces production of IFN-γ in CD4+T cells; (d) does notsubstantially bind to human CD28, CTLA-4, ICOS and BTL; (e) has nocross-reactivity with human PD-1, but has cross-reactivity with mousePD-1; and (f) is stable at least 60° C.
 43. A PD-1 binding moleculewhich competes for the same epitope with the PD-1 binding molecule ofclaim
 36. 44. An isolated nucleic acid molecule, comprising a nucleicacid sequence encoding the VHH as defined in claim
 36. 45. The isolatednucleic acid molecule of claim 44, comprising or consisting of a nucleicacid sequence as shown in any of SEQ ID NOs: 50-62.
 46. An expressionvector comprising the isolated nucleic acid molecule of claim
 44. 47. Ahost cell comprising the expression vector of claim 46, the host cell isa bacterial cell (for example, E. coli), fungal cell (for example, ayeast) or a mammalian cell.
 48. A pharmaceutical composition comprisingat least one PD-1 binding molecule as defined in claim 36 and apharmaceutically acceptable carrier.
 49. A method for preparing the PD-1binding molecule as defined in claim 36 comprising the steps of:expressing the PD-1 binding molecule as defined in claim 36 in abacterial cell, fungal cell, or a mammalian cell; and isolating the PD-1binding molecule from the host cell.
 50. A method for inhibiting orblocking the binding of PD-L1 or PD-L2 to PD-1 in a subject, comprising:administering a therapeutically effective amount of the PD-1 bindingmolecule as defined in claim 36 to the subject.
 51. A method of treatinga condition associated with PD-1 in a subject, comprising: administeringa therapeutically effective amount of the PD-1 binding molecule asdefined in claim 36 to the subject.
 52. The method of claim 51, whereinthe subject has been identified as having a disorder or a conditionlikely to respond to a PD-1 antagonist.
 53. The method of claim 52,wherein the subject has been identified as positive for presence orupregulated level of the PD-L1 or PD-L2 in a test biological sample fromthe subject.
 54. A method of treating or preventing a condition in asubject that would benefit from upregulation of immune response,comprising administering a therapeutically effective amount of the PD-1binding molecule as defined in claim 36 to the subject, wherein thesubject has upregulated expression of PD-L1 or PD-L2.
 55. A kit fortreating or diagnosing proliferative disorders such as cancers,comprising a container comprising the PD-1 binding molecule as definedin claim 36.